Journal of Traditional and Complementary Medicine xxx (xxxx) xxx

Contents lists available at ScienceDirect

Journal of Traditional and Complementary Medicine

journal homepage: http://www.elsevier.com/locate/jtcme

Formulation and evaluation of SGLT2 inhibitory effect of a polyherbal

mixture inspired from Ayurvedic system of medicine
Ankit Kumar a, Anoop Singh Negi b, Ashutosh Chauhan c, Ravindra Semwal a,
Rajnish Kumar b, Ruchi Badoni Semwal d, Randhir Singh e, f, Tushar Joshi g,
Subhash Chandra h, Sunil Kumar Joshi i, Deepak Kumar Semwal j, *
a
Research and Development Centre, Faculty of Biomedical Sciences, Uttarakhand Ayurved University, Harrawala, Dehradun, 248001, India
b
School of Pharmaceutical Sciences and Technology, Sardar Bhagwan Singh University, Balawala, 248161, Dehradun, Uttarakhand, India
c
Department of Biotechnology, Faculty of Biomedical Sciences, Uttarakhand Ayurved University, Harrawala, Dehradun, 248001, India
d
Department of Chemistry, Pt. Lalit Mohan Sharma Government Postgraduate College, Rishikesh, 249201, Uttarakhand, India
e
Department of Pharmacology, M.M. College of Pharmacy, Maharishi Markandeshwar University, Mullana, Ambala, 133207, Haryana, India
f
Department of Pharmacology, Central University of Punjab, Bathinda, 151401, Punjab, India
g
Department of Biotechnology, Kumaun University, Bhimtal, Nainital, 263136, Uttarakhand, India
h
Computational Biology & Biotechnology Laboratory, Department of Botany, Soban Singh Jeena University, Almora, 263601, Uttarakhand, India
i
Uttarakhand Ayurved University, Harrawala, Dehradun, 248001, India
j
Department of Phytochemistry, Faculty of Biomedical Sciences, Uttarakhand Ayurved University, Harrawala, Dehradun, 248001, India

a r t i c l e i n f o a b s t r a c t

Article history: Background and aim: The ingredients viz., Artemisia roxburghiana, Cissampelos pareira, Stephania glabra,
Received 31 October 2021 Drimia indica, Roylea cinerea, Tinospora sinensis and Curcuma longa of the present formulation are used to
Received in revised form treat diabetes in the Indian traditional medical system. Adopting the concept of multiple herbal mixtures
8 March 2022
for better therapeutic effects from the ancient Ayurvedic text Sarangdhar Samhita, the present study
Accepted 14 March 2022
Available online xxx
aimed to develop a polyherbal formulation (PHF) of seven herbs and to evaluate its sodium-glucose
cotransporter protein-2 (SGLT2) inhibitory effect on type 2 diabetic rats.
Experimental procedure: Streptozotocin (STZ) (60 mg/kg) and nicotinamide (NAM) (120 mg/kg) were
Keywords:
Diabetes mellitus
intraperitoneally administered to induce type 2 diabetes in Wistar rats. The animals were divided into 5
Polyherbal formulation groups viz. normal control, diabetic control, positive control (dapagliflozin at 0.1 mg/kg) and two test
Molecular docking groups (PHF at 250 and 500 mg/kg). Various parameters including blood glucose, serum glutamic pyruvic
Liver function test transaminase (SGPT), serum glutamic-oxaloacetic transaminase (SGOT), bilirubin, triglycerides and
Histopathology creatinine were measured.
SGLT2 protein Results and conclusion: The treatment with PHF (250 and 500 mg/kg) showed a significant (p < 0.05)
decrease in blood glucose levels by 56.37% and 58.17%, respectively. The levels of SGOT, SGPT and bili-
rubin were significantly reduced in PHF-fed diabetic rats. Histopathological examination revealed no
major changes in the treated groups as compared to the normal control. The molecular docking study
showed strong binding of b-sitosterol, insulanoline, warifteine, dehydrocorydalmine, taraxerol acetate,
lupeol, corydalmine and luteolin to SGLT2 protein. The present study concludes that PHF has promising
antidiabetic activity via inhibiting SGLT2 protein without showing any adverse effects.
© 2022 Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier
Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).

* Corresponding author.
E-mail addresses: [email protected], [email protected] (D.K. Semwal).
Peer review under responsibility of The Center for Food and Biomolecules, National Taiwan University.

https://doi.org/10.1016/j.jtcme.2022.03.003
2225-4110/© 2022 Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier Taiwan LLC. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: A. Kumar, A.S. Negi, A. Chauhan et al., Formulation and evaluation of SGLT2 inhibitory effect of a polyherbal mixture
inspired from Ayurvedic system of medicine, Journal of Traditional and Complementary Medicine, https://doi.org/10.1016/j.jtcme.2022.03.003
A. Kumar, A.S. Negi, A. Chauhan et al. Journal of Traditional and Complementary Medicine xxx (xxxx) xxx

1. Introduction gurjunene, a-eudesmol, curcumene, caryophyllene oxide, b-seli-

nene, germacrene D, b-eudesmol, d-cadinene, bicyclogermacrene,
Diabetes mellitus (DM), a metabolic chronic disorder, is one of artemisinin, scopoletin and quercetin have been reported from this
the major health issues that reached its alarming stage and affected plant.19 Cissampelos pareira L. (Menispermaceae) is a popular
around 500 million people worldwide irrespective of their genders. traditional remedy for ulcers, wounds, arthritis, asthma, cholera,
This number is projected to reach 700 million by the year 2045.1 diarrhoea, cancer, inflammation, snakebite, malaria, rabies and
Out of the total diabetes cases, almost 90% belong to type 2 DM diabetes.20 Its antidiabetic, antimalarial, antiparasitic, antiulcer,
which is mainly managed by different oral hypoglycemic drugs antidiarrhoeal and antivenom activities have been scientifically
including metformin, glipizide, acarbose and miglitol. However, the validated in different experimental models. Chemical analysis of its
rest 10% cases belong to type 1 DM which needs insulin therapy to roots showed the presence of bulbocapnine, corydine, dicentrine,
manage the glucose level.2 The global expenditure on diabetes and norrufescine, insulanoline, isochondrodendrine, warifteine, pelo-
its related complications was estimated to be $760 billion in the sine, cissampareine, insularine, cissampentin, hayatine, hayatinin,
adults' population of which almost 70% of expenditure belongs to dehydrodicentrine, cycleanine, reserpine, sepeerine, berberine,
the aged population of 50 years and above.3 cycleanine, corytuberine, pareitropone, cissamine, laudanosine and
Almost 90% of the existing traditional medicines worldwide are milonine.21 Stephania glabra (Roxb.) Miers (Menispermaceae) is
mainly obtained from medicinal plants and are commonly used in also used traditionally in asthma, tuberculosis, dysentery, diabetes,
their crude forms.4 The most advantage of herbal treatment is its cancer, fever and inflammation in many Asian countries.22,23
high therapeutic value with fewer adverse effects. Importantly, Similar to C. pareira, it is also rich in alkaloidal constituents like
most of the herbal remedies including spices can be used without roemerine, reticuline, palmatrubine, dehydrocorydalmine, cor-
consulting any practitioner because their use is based on traditional ydalmine, jatrorrhizine, stepharanine, capaurine, magnoflorine,
knowledge that transfers from one generation to another. It is stepholidine, n-desmethylcycleanine, tuduranine, tetrahy-
evident that the remedial values of medicinal plants are due to the dropalmatine, columbamine, laurifoline, stepharine, corynoxidine
presence of their secondary metabolites such as alkaloids, flavo- and palmatine.24 It is evident from the literature that tubers of this
noids, steroids and terpenoids.5 In addition, primary metabolites plant showed strong antidiabetic, antipsychotic, antibacterial and
like carbohydrates, proteins, vitamins and lipids also played a sig- antihypertensive effects. The bulbs of Drimia indica (Roxb.) Jessop
nificant role in human health mainly in body-building and energy (Asparagaceae) are popular for their Ayurvedic properties in car-
production. diac protection, GI disorders, asthma, leprosy, skin problems and
The ethnobotanical knowledge had reported more than 800 diabetes.25,26 Cichoriol A, urgineol A-F, anhydroscilliphaeosidin,
medicinal plants particularly useful in DM.6 Most of these plants proscillaridin A, scillaren A, scilliphaeoside, scillarenin, b-sitosterol,
are also found in India and still in traditional practice in rural areas.7 luteolin, stigmasterol, campesterol, kaempferol and quercetin are
For the past two decades, the medicinal plants are extensively the common constituents reported from this plant. Pharmacolog-
studied for their efficacy against DM for their scientific validation ical studies on the bulbs revealed anticancer, antidiabetic, antimi-
and also to find out an alternate for the existing antidiabetic drugs crobial, anti-inflammatory and bronchodilator action on different
which are known for their serious side effects.8 Based on the recent models.27
findings, these plants are capable to reduced blood glucose level via Roylea cinerea (D. Don) Baillon (Lamiaceae) is traditionally used
different mechanisms including improvement in insulin secretion for treating diabetes, malaria, stomachache and skin diseases.28 The
and glucose utilisation, activation of insulin receptors, regeneration aerial parts of this plant exhibited different pharmacological
of pancreatic b-cells and inhibition of a-glucosidase, a-amylase and properties including antidiabetic and anticancer. Its phytochemical
sodium-glucose transport protein 2 (SGLT2).9,10 In addition to using profile showed the presence of 4-methoxybenzo[b]azet-2(1H)-one,
as crude, purified molecules like berberine, curcumin, mangiferin, 3b-hydroxy-35-(cyclohexyl-50 -propan-70 -one)-33-ethyl-34-
stevioside and capsaicin obtained from these plants have also been methyl-bacteriohop-16-ene, stigmasterol, b-sitosterol, b-amyrin
investigated for their antidiabetic effect and found more effective and cetyl alcohol.29,30 Tinospora sinensis (Lour.) Merr. (Menis-
than their source extracts.11 permaceae) is well-known Ayurvedic medicine for Jaundice, fever,
Inhibition of SGLT2, a protein responsible for 80e90% glucose rheumatism, gonorrhoea and diabetes.31 Although it showed
reabsorption in the kidney, is presently one of the key targets in the diverse pharmacological activities including antidiabetic and anti-
treatment of type 2 DM and diabetes-associated complications like pyretic, its role as an anti-severe acute respiratory syndrome
diabetic kidney disease.12e14 However, possible side effects like coronavirus 2 (anti-SARS-CoV2) agent as well as a prophylaxis for
urinary tract infections, foot amputations and kidney damage by coronavirus disease (COVID-19) has gained high popularity in India
existing SGLT2 inhibitors viz. canagliflozin, dapagliflozin and and hence considered as an essential Ayurvedic drug for COVID-
empagliflozin are a matter of serious concern.15,16 Hence, herbal 19.32 Columbine, tinosporaside, jatrorrhizine, palmatine, berberine,
medicine with high efficacy and the least adverse effect can be tembeterine, tinocordifolioside, choline, tinospora acid, tinospora,
developed as a substitute for existing SGLT2 inhibitory drugs to tinosporin and tinosporide.33 Curcuma longa L. (Zingiberaceae) is
treat type 2 diabetic patients. In this direction, a formulation was traditionally used as a dietary spice and medicinal herb for different
developed with seven traditional antidiabetic herbs viz. Artemisia infections, wounds, gastrointestinal (GI) troubles and diabetes.34,35
roxburghiana (aerial parts), Cissampelos pareira (roots), Stephania Several studies reported its multiple pharmacological properties
glabra (tubers), Drimia indica (tubers), Roylea cinerea (aerial parts), such as anti-inflammatory, antioxidant, antitumor, antibacterial,
Tinospora sinensis (stems) and Curcuma longa (rhizomes) to eval- anticoagulant and antidiabetic activities.36 It contains curcumin,
uate its SGLT2 inhibitory effect. demethoxycurcumin, bisdemethoxycurcumin, turmerones, fur-
Artemisia roxburghiana Wall. ex Besser (Compositae) is used as a anodiene, ferulic acid, coumaric acid, myristicin and cyclocurcumin
traditional medicine in India for diabetes, rheumatic arthritis and as bioactive constituents.37
malarial fever.17,18 Earlier scientific studies revealed its anti- Since no established treatment is yet available for DM, research
protozoal, anthelmintic, anti-inflammatory and antidiabetic po- is continuously going on to find out a permanent solution mainly
tential. Various secondary metabolites such as taraxerol acetate, for type 2 DM. On the other hand, various earlier researches
lupeol, friedelan-3b-ol, friedelin, betulinic acid, betulin, apigenin, revealed that a single herb is sometimes insufficient to achieve the
artemisinin, apigenin-7,4-dimethyl ether, a-selinene, a-copaene, a- required medicinal effect. However, the combination of more than
2
A. Kumar, A.S. Negi, A. Chauhan et al. Journal of Traditional and Complementary Medicine xxx (xxxx) xxx

one herb, called polyherbalism, produces enhanced medicinal corresponding pure standard. The identified metabolites were
properties as well as less toxicity, perhaps due to the synergistic further compared with the available literature on the plant.
effect,38 and this remains the actual challenge in the development
of polyherbal formulation. The herbs used in the present study are 2.4. Preparation of polyherbal formulation
used in traditional medicine either as a single herb or in the form of
a formulation.8,39e41 Hence, the present research aimed to develop The polyherbal formulation (PHF) was prepared by mixing all
a polyherbal formulation inspired from Ayurveda to treat diabetes hydroalcoholic extracts in a definite ratio by following Bhavprakash
via inhibiting SGLT2 protein. Nighantu, an Ayurvedic classical text.26 The PHF contained 7.42%
A. roxburghiana (aerial parts), 22.26% C. pareira (roots), 7.42%
2. Materials and methods S. glabra (tuber), 0.15% D. indica (tuber), 7.42% R. cinerea (aerial
parts), 29.69% T. sinensis (stems) and 25.64% Curcuma longa
2.1. Collection and authentication of plant material (rhizomes).

The plant samples were collected from the different localities of 2.5. Experimental animals
the Garhwal and Kumaon regions of Uttarakhand Himalaya. The
authentication of the plants was done by the Botanical Survey of Wistar rats with 80e120 g weight were selected for the study
India, Dehradun and R&D Centre, Uttarakhand Ayurved University, irrespective of their sex. The animal studies were conducted at
Dehradun. The accession/voucher numbers 118189, 118595, 118596, M.M. College of Pharmacy, Ambala with ethical approval No.
118597, UAU211, UAU229 and UAU253 were assigned to Artemisia MMCP/IAEC/55/2019. All rats were acclimatized to the laboratory
roxburghiana Wall. ex Besser, Drimia indica (Roxb.) Jessop, Cissam- condition for one week before starting the study. The general
pelos pareira L., Stephania glabra (Roxb.) Miers, Tinospora sinensis behaviour, body weight, and feed-water intake of the rats were
(Lour.) Merr., Roylea cinerea (D.Don) Baill. and Curcuma longa L., observed during the acclimatization period. The animals were
respectively. maintained as per Control and Supervision of Experiments on An-
imals (CPCSEA) guidelines, and experimentation was done as per
2.2. Preparation of extracts the approved protocol. The rats were kept on a permitted diet and
water ad lib during the study.
The freshly collected plant parts were thoroughly washed with
plenty of tap water to remove impurities and put in the hot air oven 2.6. Acute toxicity study
at 50  C for 48 h with occasional turns upside down. The dried and
coarsely powdered material was soaked with distilled water and The rats were divided into 2 groups of 6 rats per group irre-
absolute ethanol (1:1) for 72 h (three times, each for 24 h) for spective of their sex to assess the acute toxicity as per Organisation
complete extraction. The solvent was removed using a vacuum for Economic Co-operation and Development (OECD) guidelines.42
rotary evaporator (Equitron, India) with bath temperature at 50  C Group 1 served as a control and provided a normal diet whereas
to obtain a dry extract which was then stored at 4  C until further group 2 is given a single oral dose of PHF at 2000 mg/kg, body
use. weight and also allowed a normal diet. All rats were monitored
continuously every hour for the first day and then every day for
2.3. GC-MS/MS metabolomics analysis of extracts fourteen days to assess the different parameters including skin, fur,
eyes, respiratory pattern, salivation, diarrhoea, urination, tremors,
GC-MS analysis of the extract was performed on Agilent 7890B ptosis, relaxation, gait, posture, lethargy, sleep, coma and food/
gas chromatograph (Agilent Technologies, CA, USA) coupled with water intake pattern together with other behavioural changes.
an Agilent 5977B mass detector. The sample was injected into GC- However, the body weight of the rats was measured after every 4
MS by an automatic sampler (CN1700443 3 series). HP-5 MS col- days.43
umn (5% phenyl methyl polysiloxane; 30 m  0.25 mm i.d. x
0.25 mm) and helium as a carrier gas were used for metabolites 2.7. Induction of diabetes
separation. For GC-MS analysis, the extract was dried in an
Eppendorf concentrator and resuspended in 100% methanol and The diabetes was induced in overnight fasted rats by a single
further dried in the concentrator, and then directly derivatized with intraperitoneal injection of 60 mg/kg of STZ (Sisco Research Labo-
70 mL of N-methyl-N-(trimethylsilyl) trifluoroacetamide for 60 min ratories, India), freshly prepared in 0.1 M citrate buffer (pH 4.5),
at 37  C in a 1.5 mL centrifuge tube followed by centrifugation at 15 min after the intraperitoneal administration of 120 mg/kg of
13000 rpm for 10 min. The resulting supernatant was taken out and NAM (Sisco Research Laboratories, India), which was prepared in
directly analyzed by the GC-MS system. The injection volume was saline solution. The diabetes was confirmed by the elevated blood
1 mL with splitless mode. The injector temperature was set at glucose level determined at 72 h after the STZ/NAM administration.
280  C. The oven temperature was initially set at 80  C for 2 min, The rats with blood glucose levels of more than 250 mg/dL were
then ramped to 220  C at a rate of 10  C/min without any hold and considered diabetic and used for the present study.44
further increased at 310  C at the rate of 20  C/min held for 10 min
and with a solvent delay of 5 min. The column flow rate was 1 mL/ 2.8. Experimental design
min. The conditions for the operation of the mass spectrometer
were set as follows: ion source temperature 230  C, MS Quad The rats were divided into five groups (n ¼ 6) in which the first
temperature 150  C, electron energy (70eV) and scanning range of group comprised of non-diabetic rats whereas the rest of the
m/z, 25e1000 amu. Metabolites were identified by matching the groups included diabetic rats. The normal control group (G1NC)
mass spectra of target metabolite (3:1 signal to noise ratio) with the and diabetic control group (G2DC) did not receive any treatment
NIST-17 mass spectral library. Metabolite identity was reported but were allowed free access to distilled water. The third group
only when the matching value of the mass spectra comparison was (G3S) received standard drug dapagliflozin orally at the dose of
more than 80%, and an increase in the area of the corresponding 0.1 mg/kg/day in distilled water whereas the fourth group (G4T1)
peak was observed when spiking the sample with the and fifth group (G5T2) received PHF at the oral doses of 250 and
3
A. Kumar, A.S. Negi, A. Chauhan et al. Journal of Traditional and Complementary Medicine xxx (xxxx) xxx

500 mg/kg/day, respectively in distilled water for 28 days. The ligand complexes, including hydrogen bonds and other bonds, were
doses of PHF were determined based on acute toxicity study. analyzed and depicted using discovery studio software.

2.9. Biochemical analysis 2.14. Statistical analysis

The blood samples were collected from retro-orbital plexus All the values are shown as means ± standard error of the mean
using ether anaesthesia.45 Blood collected into the Clot Activator (SEM). Statistical analyses were performed using one-way ANOVA
tubes or Fluoride tubes (for glucose test) was stored at 4  C for 3 h followed by Tukey's test and analyzed using the GraphPad Prism 8
and then centrifuged at 3000 revolutions per minute for 10 min software. The p values equal to or less than 0.05 were considered
using MiniSpin Plus Microcentrifuge (Eppendorf, Germany) to statistically significant.
separate the serum.46 The isolated serum samples were used to
measure the levels of fasting blood glucose (FBG), SGPT, SGOT, total 3. Results
bilirubin, cholesterol, triglyceride, creatinine, uric acid, urea and
lactate dehydrogenase (LDH). All the parameters were assessed 3.1. Identification of metabolites
routinely on the 0th, 7th, 14th, 21st and 28th day using Standard
Assay Erba Diagnostic Kits (Erba Semi-Auto Biochemistry Analyzer, Based on the GC-MS/MS analysis, most intense peaks with highest
UK). In addition, body weight of rats was also measured regularly peak width were characterized from each methyl derivatized extract.
using digital weighing balance. The results revealed 2-pyrrolidinone; L-arabitol; a-lyxopyranose;
palmitic acid; bisphenol A, bis(trimethylsilyl) ether and acethy-
2.10. Isolation of organs and their histopathology drazide, N2-[4-(thiitan-3-yloxy)benzylideno]- as main constituents
of the extract of C. pareira. A. roxburghiana extract was found to
At the end of the 28th day, the rats were painlessly sacrificed by contain protocatechoic acid; caffeic acid; arachidonic acid and azelaic
cervical dislocation.47 The liver, kidney and pancreas of each rat acid. Similarly, 2-methylpropanoic acid; 2-hexenedioic acid, bis(-
were carefully removed for histopathological examination. The trimethylsilyl) ester; D-tagatofuranose, pentakis(trimethylsilyl)
organs were washed with normal saline and preserved in a 5% ether; acetin, bis-1,3-trimethylsilyl ether and levoglucosan were
formalin solution (Rankem, India) at room temperature until characterized from D. indica extract. In case of T. sinensis methyl
further use.48 The histopathological examination of isolated organs derivatized extract, 2-butenedioic acid; 5a-androstan-3b-ol, O-
was performed by Dr. Lal Pathlabs Ltd., New Delhi, India using a methyl-; ((11,11-dimethyl-8-methylenebicyclo[7.2.0]undecan-4-yl)
microscope at 10x, 20x, 40x and 100x magnifications. methoxy)trimethylsilane; 2,5-dichlorohydroquinone; ethane-
sulfonic acid, 2-[(trimethylsilyl)oxy]-, trimethylsilyl ester; syringar-
2.11. Molecular modelling for SGLT2 protein esinol and b-epinephrine were found as major constituents. However,
the extract of S. glabra comprised of cyclopropane, 1-(1-
Due to the unavailability of three dimensional (3-D) structure of hydroxyethyl)-1-(diethylphosphonyl)-2-methylene-; 5a-androstan-
SGLT2 of Homo sapiens, the sequence of SGLT2 was retrieved from 3b-ol, O-methyl-; pyridine-3-sulfonamide, 2,6-dichloro-4-methyl-N-
NCBI (accession number- NP_003032.1), and the 3D structure was cyclohexyl-; 6H-dibenzo[a,g]quinolizine, 5,8,13,13a-tetrahydro-
predicted using the “Easy Modeller” homology modelling mod- 2,3,9,10-tetramethoxy-; 11-hydroxyetiocholanolone (3a,5a,11b) and
ule.49 The generated 3D structures of SGLT2 were subjected to 2-{2-oxo-4-[(trimethylsilyl)oxy]pyrrolidin-1-yl}-N-(trimethylsilyl)
further optimization, which includes minimization, side chain as acetamide as main constituents.
well as loop refinement. The optimized structure was submitted
into the PDBsum server of protein data bank (PDB)50 to validate the 3.2. Acute toxicity
modelled structure.
All the animals treated with a single oral dose of PHF at
2.12. Active site confirmation 2000 mg/kg showed normal behavioural, motor, and neuronal
functions. No mortality was observed during the experimental
After modelling, the computed atlas of surface topography of period up to 14 days. The monitoring of skin, fur, eyes, respiratory
proteins (CASTp) webserver was used to find out the active side in pattern, autonomic nervous system (ANS) characteristics (i.e.,
protein and locate delineating, and measuring geometric and to- salivation, diarrhoea and urination) and central nervous system
pological properties of the modelled SGLT2 structure.51 The CASTp (CNS) characteristics (i.e., tremors, ptosis, relaxation, gait and
3.0 predicted many active amino acid residues which may present posture) in the treated rats remained unaffected. Moreover, the
in the active site and are responsible for protein-ligand interaction. water and feed intake pattern of the rats was regular and consistent
during the study period. The changes in body weight of the treated
2.13. Molecular docking and visualization group did not record any substantial changes when compared with
the control. Hence, based on the acute toxicity outcome, PHF can be
Docking was performed to obtain a population of possible orien- considered safe up to 2000 mg/kg body weight.
tations and conformations for the ligand at the binding site by using
InstaDock open-source software.52 This software performs the pre- 3.3. Effect on body weight
diction of the bound conformations based on the binding affinity. The
grid centre for docking analysis was set to X ¼ 25.462, Y ¼ -43.18, The weekly measurement of body weight showed no significant
and Z ¼ 61.307, and the dimensions of the grid box were set as changes in diabetic control and standard group. However, the
84  110  99 Å having a spacing of 0.375 Å between the grids points. formulation with a higher dose showed a slight increase in the body
The virtual screening of compounds was conducted by rigid molec- weight of diabetic rats when compared with the control group. The
ular docking in the active site of SGLT2 protein. A total of 170 com- average body weight of the animals treated with 500 mg/kg was
pounds for molecular docking study were selected from our previous recorded to be 100 ± 8.003 g on the 28th day which was increased
reports on the individual herbs of the present PHF and other antidi- from 87 ± 7.278 on a basal day whereas, at a dose of 250 mg/kg, the
abetic agents.11,17,19,21,24,27 Molecular interactions between protein- body weight increased to 100 ± 3.921 from 94 ± 4.416.
4
A. Kumar, A.S. Negi, A. Chauhan et al. Journal of Traditional and Complementary Medicine xxx (xxxx) xxx

Fig. 1. Effect of PHF on blood glucose level of rats. G1NC (normal control group), G2DC (diabetic control group), standard group (G3S), test group 1 (G4T1) and test group 2 (G5T2);
(*) shows significance level with p value of <0.05 when compared to G2DC.

3.4. Effect on blood glucose level decrease was noted till the end of the experiment (Table 1). Simi-
larly, total bilirubin levels were increased in the diabetic control
After confirmation of hyperglycemia (fasting glucose level group during the entire experimental period while the changes
>250 mg/dL), the rats were treated with PHF and standard drug. measured in the treated groups were found insignificant till 14 days
The results (Fig. 1) showed that blood glucose levels of all treated and significantly increased thereafter. Hence, it can be said that the
groups were significantly (p < 0.05) decreased on the 7th day of PHF has long-term significant effects on the levels of SGPT, SGPT
treatment and remains effective till the 28th day when compared and total bilirubin in STZ-NAM-induced diabetic rats.
to the normal control group. The PHF showed 56.37% and 58.17%
reduction in glucose levels after 28 days at the doses of 250 and
500 mg/kg, respectively whereas 53.90% reduction was recorded 3.6. Effect on lipid profile
with 0.1 mg/kg of dapagliflozin.
The levels of cholesterol and triglycerides in the serum of
3.5. Effect on liver function experimental rats were measured to evaluate the effect of PHF on
lipid profile. Their levels were found higher in the diabetic control
The STZ-NAM-induced hyperglycemia significantly increased group when compared to the normal control group. The results
the SGPT and SGOT levels in rats from the 7th to the 28th day. (Table 2) revealed a significant (p < 0.05) change after two weeks in
However, there were no significant changes occurred in the levels the levels of cholesterol and triglycerides in diabetic rats treated
of SGPT and SGOT in the standard and PHF-treated groups for up to with both PHF and dapagliflozin when compared to the diabetic
14 days. Interestingly, 14th day onwards, a significant (p < 0.05) control group.

Table 1
Effect of PHF on liver function in experimental rats.

Day G1NC G2DC G3S G4T1 G5T2

SGPT (U/L)
Basal (0) Day 28.40 ± 4.69 32.10 ± 5.35 34.69 ± 3.25 32.25 ± 6.16 37.28 ± 0.49
7th Day 31.54 ± 5.77 41.77 ± 9.79 43.30 ± 7.27 36.86 ± 5.29 47.14 ± 8.92
14th Day 31.45 ± 5.05 76.02 ± 16.54 87.51 ± 12.30 54.16 ± 7.80 58.75 ± 7.68
21st Day 33.78 ± 4.74 119.53 ± 8.44 60.76 ± 5.13* 47.28 ± 15.81* 53.51 ± 13.84*
28th Day 32.49 ± 3.36 98.64 ± 11.80 40.28 ± 15.03* 40.39 ± 6.84* 48.31 ± 7.45*
SGOT (U/L)
Basal (0) Day 89.10 ± 5.60 92.82 ± 10.97 103.47 ± 12.56 101.48 ± 14.86 89.09 ± 4.63
7th Day 86.95 ± 7.84 129.61 ± 6.72 119.05 ± 33.85 116.36 ± 8.33 112.38 ± 3.40
14th Day 92.82 ± 10.37 169.17 ± 8.87 140.74 ± 8.26 137.20 ± 7.92 113.84 ± 10.44*
21st Day 86.03 ± 12.70 147.52 ± 15.57 138.70 ± 13.21 123.69 ± 13.05 101.70 ± 5.89*
28th Day 81.33 ± 10.25 181.20 ± 7.29 119.72 ± 9.46* 115.34 ± 14.68* 118.35 ± 8.37*
Total bilirubin (mg/dL)
Basal (0) Day 0.66 ± 0.14 0.79 ± 0.16 0.62 ± 0.10 0.83 ± 0.09 0.88 ± 0.10
7th Day 0.87 ± 0.35 0.94 ± 0.16 0.86 ± 0.22 0.91 ± 0.30 0.92 ± 0.13
14th Day 0.68 ± 0.10 0.82 ± 0.22 0.73 ± 0.18 0.89 ± 0.09 0.84 ± 0.09
21st Day 0.88 ± 0.06 0.94 ± 0.09 0.53 ± 0.07* 0.71 ± 0.08* 0.77 ± 0.23*
28th Day 0.87 ± 0.08 1.03 ± 0.22 0.69 ± 0.46* 0.79 ± 0.30* 0.88 ± 0.32*

G1NC (normal control group), G2DC (diabetic control group), standard group (G3S), test group 1 (G4T1) and test group 2 (G5T2); (*) Shows significance level with p value of
<0.05 when compared to G2DC.

5
A. Kumar, A.S. Negi, A. Chauhan et al. Journal of Traditional and Complementary Medicine xxx (xxxx) xxx

Table 2
Effect of PHF on lipid profile of experimental rats.

Day G1NC G2DC G3S G4T1 G5T2

Cholesterol (mg/dL)
Basal (0) Day 36.64 ± 8.83 40.52 ± 4.79 45.15 ± 3.46 39.81 ± 6.90 38.46 ± 9.06
7th Day 41.56 ± 4.77 43.06 ± 1.68 46.76 ± 5.40 50.5 ± 10.98 43.79 ± 4.98
14th Day 41.37 ± 5.31 83.28 ± 9.65 59.85 ± 11.49* 44.13 ± 11.69* 45.39 ± 7.64*
21st Day 37.51 ± 4.81 74.81 ± 15.99 59.86 ± 11.98 44.7 ± 5.27* 48.00 ± 6.92*
28th Day 36.79 ± 12.23 80.05 ± 4.72 56.67 ± 7.29* 40.9 ± 3.59* 45.02 ± 11.02*
Triglycerides (mg/dL)
Basal (0) Day 37.61 ± 5.32 37.13 ± 2.89 34.81 ± 8.94 40.61 ± 7.00 47.11 ± 57.55
7th Day 37.81 ± 14.86 42.56 ± 9.24 48.07 ± 13.58 43.74 ± 7.39 42.87 ± 3.27
14th Day 44.58 ± 18.51 45.84 ± 10.95 42.30 ± 17.55 49.62 ± 22.68 41.13 ± 17.62
21st Day 40.68 ± 15.58 80.24 ± 22.12 48.47 ± 21.23* 42.55 ± 30.34* 48.20 ± 12.40*
28th Day 47.92 ± 7.80 86.86 ± 13.15 42.06 ± 16.00* 44.47 ± 12.43* 40.74 ± 9.17*

G1NC (normal control group), G2DC (diabetic control group), standard group (G3S), test group 1 (G4T1) and test group 2 (G5T2); (*) Shows significance level with p value of
<0.05 when compared to G2DC.

Table 3
Effect of PHF on renal function of experimental rats.

Day G1NC G2DC G3S G4T1 G5T2

Creatinine (mg/dL)
Basal (0) Day 0.69 ± 0.02 0.68 ± 0.02 0.70 ± 0.05 0.73 ± 0.03 0.67 ± 0.06
7th Day 0.74 ± 0.14 0.73 ± 0.10 0.73 ± 0.19 0.76 ± 0.10 0.69 ± 0.13
14th Day 0.66 ± 0.05 0.75 ± 0.16 0.70 ± 0.04 0.74 ± 0.05 0.65 ± 0.08
21st Day 0.84 ± 0.17 0.89 ± 0.19 0.80 ± 0.13 0.82 ± 0.07 0.74 ± 0.12
28th Day 0.70 ± 0.02 0.85 ± 0.07 0.70 ± 0.02 0.68 ± 0.05 0.68 ± 0.09
Uric acid (mg/dL)
Basal (0) Day 2.26 ± 0.38 2.62 ± 0.22 2.21 ± 0.65 2.46 ± 1.12 2.44 ± 0.48
7th Day 3.58 ± 1.90 3.70 ± 0.79 2.81 ± 1.06 2.90 ± 1.83 3.45 ± 0.72
14th Day 2.89 ± 0.78 3.38 ± 0.50 2.91 ± 0.94 2.23 ± 0.49 2.93 ± 0.44
21st Day 2.87 ± 0.29 3.46 ± 0.87 2.48 ± 1.08 3.21 ± 1.09 2.91 ± 0.48
28th Day 2.19 ± 0.52 2.58 ± 0.61 2.48 ± 0.39 2.18 ± 0.53 2.74 ± 1.10
Urea (mg/dL)
Basal (0) Day 82.89 ± 14.28 70.40 ± 17.99 85.49 ± 12.67 81.87 ± 19.22 80.27 ± 13.20
7th Day 86.12 ± 11.84 80.96 ± 3.45 111.40 ± 3.67 91.87 ± 8.97 82.33 ± 6.49
14th Day 83.47 ± 6.36 80.10 ± 8.80 90.43 ± 3.54 97.93 ± 1.98 88.37 ± 6.71
21st Day 82.47 ± 3.18 88.87 ± 4.01 96.71 ± 8.56 97.81 ± 6.71 85.80 ± 5.69
28th Day 85.87 ± 18.68 118.58 ± 18.12 99.32 ± 11.11 97.55 ± 14.52 92.34 ± 7.60

G1NC (normal control group), G2DC (diabetic control group), standard group (G3S), test group 1 (G4T1) and test group 2 (G5T2); (*) Shows significance level with p value of
<0.05 when compared to G2DC.

3.7. Effect on renal function blood sinusoids in all the groups. Mild sinusoidal congestion, as
well as congestion of central and portal vein, was seen in G2DC and
The present study showed that dapagliflozin and PHF did not G5T2 groups similar to the G1NC group, whereas mild congestion
produce any significant changes in the levels of creatinine, uric acid of sinusoids was seen in the G4T1 group. Hepatocytes were poly-
and urea in serum of diabetic rats. A slight increase in the levels of hedral in shape with slightly vacuolated granular cytoplasm and
urea was although recorded in all treated groups but it was vesicular nuclei in all the groups similar to the normal control
comparatively lower than that of the diabetic control group group. Focal area of mild degenerative changes with swollen he-
(Table 3). patocytes and some cells with karyolitic nuclei were present in
G5T2 similar to the G1NC group, whereas the focal area of mild
3.8. Effect on LDH level degenerative changes with mild steatosis and some cells with
karyolitic nuclei were present in G4T1. Blood sinusoids separating
The diabetic control group showed a significant increase in their the hepatic cords lined by endothelial cells and kupffer cells were
LDH level on the 28th day when compared to the normal control seen in the G3S group only. Bi-nucleated hepatocytes with no
group. The treated groups also showed an increase in the LDH level infiltration or granuloma were also seen in all the groups (Fig. 3).
but this change was found insignificant (Fig. 2). There was no sig-
nificant difference in the effect of PHF (at 250 and 500 mg/kg) and 3.9.2. Histopathology of pancreas
dapagliflozin (0.1 mg/kg). The section of G1NC showed normal architecture of the
pancreas with very mild degenerative changes. The exocrine
3.9. Histopathology of isolated organs pancreas is composed of closely packed acinar cells and arranged
into small lobules. Pancreatic lobules are separated by intact intra-
3.9.1. Histopathology of liver lobular and interlobular connective tissue septa. The islet cells were
Microscopic examination of sections revealed the normal his- seen interspersed between the acinar cells. Islets appeared lightly
tological structure of hepatic lobules in both treated and untreated stained than the surrounding acinar cells. However, the histopa-
animals including the normal control group. The hepatocytes were thology of the pancreas of G2DC revealed moderate to high
arranged in cords radiating from the central vein and separated by degenerative changes in both exocrine and endocrine components.
6
A. Kumar, A.S. Negi, A. Chauhan et al. Journal of Traditional and Complementary Medicine xxx (xxxx) xxx

Fig. 2. Effect of PHF on LDH level in experimental rats. G1NC (normal control group), G2DC (diabetic control group), standard group (G3S), test group 1 (G4T1) and test group 2
(G5T2); the results were found insignificant when compared to G2DC.

Fig. 3. Histopathology of right kidney (KR), left kidney (KL), pancrease (P) and liver (L) of experimental rats. (Y): Mild sinusoidal congestion; (,): mild degenerative changes; (⌂):
moderate degenerative changes; (B) high degenerative changes; ⃝() depletion of beta cells; (D): desquamation of epithelial linings; G1NC (normal control group), G2DC (diabetic
control group), standard group (G3S), test group 1 (G4T1) and test group 2 (G5T2); the results were found insignificant when compared to G2DC.

Acinar cells were swollen and small vacuoles were observed in hand, all the treated groups showed mild degenerative changes in
acinar cells. Interlobular ducts were lined with flattened epithe- both exocrine and endocrine components similar to G1NC. In
lium. Some lobules showed depletion of islet b-cells. On the other addition, a few acinar cells swollen with small vacuoles were

7
A. Kumar, A.S. Negi, A. Chauhan et al. Journal of Traditional and Complementary Medicine xxx (xxxx) xxx

observed. Interlobular ducts were lined with flattened epithelium.

A small number of lobules in G3S, G4T1 and G5T2 showed deple-
tion of islet b-cells (Fig. 3).

3.9.3. Histopathology of right kidney

Microscopic examination of sections of the right kidney
revealed mild haemorrhages present in between intertubular
spaces in all the groups. Mildly congested blood vessels were also
seen in all the groups. There are mild to moderate degenerative
changes of epithelial linings of tubules seen in G2NC whereas all
treated groups showed mild degenerative changes similar to the
normal control group. Some tubules show desquamation of
epithelial linings in G2DC and G3S groups. However, focal inter-
stitial inflammatory mononuclear cell infiltrate were seen in G2DC
but not in G3S. Similar to G1NC, no granuloma was seen in the rats
treated with standard and PHF (Fig. 3).

3.9.4. Histopathology of left kidney

Microscopic examination of the left kidney showed most his-
tological features similar to the right kidney. G5T2 showed focal
mild intertubular haemorrhages. An infiltration of polymorph cells
was present in surrounding fibro-fatty tissues in G5T2. Small cystic
areas were only present in the G4T1 group. In the G3S group, some
of the tubules showed desquamation of epithelial linings and mild
expansion of the glomerular cavity similar to the normal control
group. Additionally, no infiltration of inflammatory cells or granu-
loma was seen in the G3S group. G1NC and G2DC showed focal
interstitial inflammatory mononuclear cell infiltrates. Focal tubular
basophilia, nuclear crowding, and thickened basement membranes
were also present in the G2DC group. In the treatment group G4T1,
tubules showed necrosis of epithelial linings (Fig. 3).

3.10. Homology modelling and active site confirmation Fig. 4. Active sites of SGLT2 protein.

The SGLT2 protein sequence of Homo sapiens was retrieved from

NCBI, and a basic local alignment search tool-protein (BLASTp) was active compounds with their docking scores, natural sources, 2D
used to identify homologous templates from the PDB database. and 3D molecular docking interactions with SGLT2 protein are
BLASTp showed similarities of the SGLT2 query with the crystal shown in Fig. 5.
structure of secretogranin-2 (SGC2) of Vibrio parahaemolyticus (PDB
ID 2XQ2 A, 3DH4 and 2XQ2 B), and these structures share 69%, 64% 4. Discussion
and 63% identity respectively query cover. After that, Easy Modeller
software was used for the prediction of the 3D structure of SGLT2 Based on the global research outcomes to date, it is understood
using four structures as templates. The 3-D model of protein also that DM is incurable, although it can be managed with the help of
showed 90.5% of the residues in the allowed region of the Ram- selected medications including modern and traditional medicine.53
achandran plot. It demonstrates the stereochemical reliability of The treatment of DM with herbal medicine is considered a safer
the homology template of SGLT2 protein. approach which has been practised in different traditional systems
The active site (Fig. 4) residue numbers of SGLT2 of Homo sapiens of medicine since ancient times.19 Moreover, the treatment with
are 32, 33, 36, 37, 40, 41, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, multiple herbs showed better effects with lesser toxicity than a
56, 57, 58, 59, 60, 61, 62, 63, 65, 66, 69, 70, 72, 73,74, 75, 76, 77, 78, single herb, perhaps due to the synergistic effect of different con-
79, 80, 83, 84, 95, 96, 98, 99, 102, 123, 124, 125, 126,127,129, 130, 136, stituents present in these herbs.54,55 In the past couple of years,
139, 142, 143, 145, 146, 149, 150, 153, 154, 157, 158, 161, 184, 187, 188, tremendous work has been done on the polyherbal formulations
189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 203, and many of them were found effective in controlling hyperglyce-
204, 205, 208, 209, 283, 286, 287, 289, 290, 291, 294, 295, 296, 297, mia in different experimental models.56e58
300, 301, 304, 320, 321, 324, 329, 387, 388, 389, 390, 391, 392, 393, The evaluation of toxicity of a plant sample is a key step for
395, 396, 397, 398, 399, 400, 402, 403, 404, 406, 407, 411, 412, 414, conducting pharmacological studies in higher models. Such studies
415, 416, 417, 418, 419, 420, 422, 423, 424, 426, 427, 430, 452, 453, give initial information about the safety profile of a drug candidate
456, 460, 463, 464, 467, 471, 552, 553, 659, 662, 663, 666, 667, 670, and also help in deciding the doses for in vivo studies.59 In the
671, 672. Molecular docking was performed with SGLT2 of Homo present study, PHF was evaluated for its acute toxicity in rats and
sapiens, specifying the active site using the amino acids area. based on the outcome of this experiment, the doses for antidiabetic
activity were determined. Since the highest doses at 2000 mg/kg
3.11. Molecular docking did not produce any toxic effect in rats up to 14 days, the oral LD50
of PHF can be considered to be higher than 2000 mg/kg.
Phytochemicals of plants were screened by molecular docking For the past couple of years, a combination of STZ and NAM is most
against the SGLT2 receptor. Out of 170 compounds, b-sitosterol and accepted to develop type 2 DM because STZ causes b-cell damage
insulanoline showed the highest binding energy. The top eight while NAM partially protects the insulin-secreting cells against STZ
8
A. Kumar, A.S. Negi, A. Chauhan et al. Journal of Traditional and Complementary Medicine xxx (xxxx) xxx

Fig. 5. 2D and 3D molecular docking interactions of bioactive compounds with SGLT2 protein. DS (docking score - kcal/mol), BS (botanical source).

and hence, protects them from permanent damage.60 The present The increased levels of lipids, mainly cholesterol and tri-
study found STZ-NAM-induced diabetic model as the most suitable to glycerides, in diabetic patients are risk factors for diabetes-
evaluate the SGLT2 inhibitory effect in rats. The administration of STZ associated cardiovascular diseases.62 It is evident that the risk of
and NAM produces a significant elevation in the glucose levels of developing hypertension is higher in DM patients than in others.63
experimental rats when compared to the normal group. Moreover, chronic diabetes plays a key role in developing vascular
The reduction in blood glucose level is the first priority in diseases by increasing the formation of advanced glycation end-
developing an antidiabetic drug because hyperglycemia can cause products (AGEs), activation of the receptor for advanced glycation
various complications including retinopathy, nephropathy and endproducts (RAGE), oxidative stress and inflammation.64 The re-
neuropathy.61 In the present study, the treatment of PHF (250 and sults obtained from the present study revealed a significant change
500 mg/kg) significantly (p < 0.05) decreased the elevated blood in the levels of total cholesterol and triglycerides both in test groups
glucose levels in STZ-NAM induced diabetes rats within one week and positive control. The maximum reduction was recorded on the
and this effect was also recorded during the entire experimental 28th day of the experiment.
period when compared with the diabetic control group. The study Similarly, the increased levels of SGPT, SGOT and bilirubin due to
also found that the effect of PHF was comparable to that of the liver injury are common conditions mainly in chronic diabetes. This
positive control group at the end of the 28th day. Moreover, body condition can also affect the metabolism of lipids, carbohydrates
weight is an important parameter to be monitored regularly in the and proteins and hence causes fatty liver. Moreover, developing
event of diabetes. However, in the present study, no significant liver cirrhosis, hepatocellular carcinoma and oxidative stress were
changes were recorded in body weight of experimental rats. also recorded in the patients with chronic DM.65 In the present

9
A. Kumar, A.S. Negi, A. Chauhan et al. Journal of Traditional and Complementary Medicine xxx (xxxx) xxx

study, the PHF significantly decreased the levels of SGPT and SGOT Experiments on Animals (CPCSEA) guidelines, and experimentation
enzymes after the 14th day. Moreover, total bilirubin levels were was done as per the approved protocol.
increased in the diabetic control group during the entire experi-
mental period while the changes measured in the treated groups Authors’ contribution
were found significant when compared to G2DC. Hence, it can be
said that the PHF has a significant effect on the levels of SGPT, SGPT DKS: conceptualized and supervised the work, edited and
and total bilirubin in STZ-NAM-induced diabetic rats and as a result, reviewed the manuscript; AK, ASN, RK and RSD: developed the
it might play a potential role in the hepatoprotective activity. On the formulation and conducted the animal experiments; TJ and SC:
other hand, elevated levels of creatinine, urea and uric acid in the conducted the molecular docking studies; AK and RS: prepared the
blood are indications of kidney problems. The present study did not first draft of the manuscript; RS and AC: statistically analyzed the
find a significant change in the levels of these components when data: RBS and SKJ: edited and reviewed the manuscript.
compared to the control group.
The histopathological examination of isolated organs was done Declaration of competing interest
to find any internal toxic sign in the treated animals. The sample
can be considered safe if its histology matches with normal ani- The authors declared no conflict of interest.
mals. This study showed that PHF does not cause any harmful effect
on the experimental rats when compared to diabetic rats. The re-
Acknowledgement
sults of PHF for liver and kidney function were supported by his-
topathological analysis. Moreover, the pancreas was also found
This research was funded by National Medicinal Plants Board,
unaffected after the 28-days of in vivo study. Overall, the formula-
Ministry of AYUSH, Govt. of India (Grant No. Z.18017/187/CSS/R&D/
tion was found effective and safe for further use.
UK-01/2017-18-NMPB-IV A).
Dapagliflozin, a synthetic antidiabetic drug, has the potential to
reduce blood glucose specifically by inhibiting SGLT2 protein in the
renal tubule to block glucose reabsorption. In addition to reducing References
HbA1c level, this drug also helps in reducing body weight.66 1. IDF. IDF Diabetes Atlas. ninth ed. International Diabetes Federation; 2019.
However, previous studies indicate that the SGLT2 inhibitors may https://www.diabetesatlas.org/en/.
produce adverse effects on the skeleton which results in the 2. Chaudhury A, Duvoor C, Reddy Dendi VS, et al. Clinical review of antidiabetic
drugs: implications for type 2 diabetes mellitus management. Front Endocrinol.
weakening of bones in diabetic patients.67 The present study used
2017;8:6. https://doi.org/10.3389/fendo.2017.00006.
dapagliflozin as a standard drug to compare the antidiabetic effect 3. Williams R, Karuranga S, Malanda B, et al. Global and regional estimates and
of PHF. The results showed that the values of most of the param- projections of diabetes-related health expenditure: results from the interna-
eters studied for PHF were comparable to the standard drug. Hence, tional diabetes federation diabetes atlas. ninth ed. Diabetes Res Clin Pract.
2020;162:108072. https://doi.org/10.1016/j.diabres.2020.108072.
in view of the potential glucose-lowering effect and safety profile, 4. Sofowora A, Ogunbodede E, Onayade A. The role and place of medicinal plants
the PHF was also evaluated for its SGLT2 inhibitory effect via in silico in the strategies for disease prevention. Afr J Tradit, Complementary Altern Med.
method using major bioactives of the herbs present in PHF. 2013;10(5):210e229. https://doi.org/10.4314/ajtcam.v10i5.2.
5. Hounsome N, Hounsome B, Tomos D, Edwards-Jones G. Plant metabolites and
Nowadays, molecular docking studies are most popular among nutritional quality of vegetables. J Food Sci. 2008;73(4):R48eR65. https://
the scientific community for the early inclusion of pharmacoki- doi.org/10.1111/j.1750-3841.2008.00716.x.
netics consideration in the process of novel drug discovery with the 6. Bilal M, Iqbal MS, Shah SB, Rasheed T, Iqbal HMN. Diabetic complications and
insight into antidiabetic potentialities of ethno-medicinal plants: a review.
help of advanced software.68 The present study identified eight Recent Pat Inflamm Allergy Drug Discov. 2018;12(1):7e23. https://doi.org/
bioactive phytochemicals including b-sitosterol and insulanoline 10.2174/1872213X12666180221161410.
which showed strong binding to the SGLT2 protein with a docking 7. Saxena A, Vikram NK. Role of selected Indian plants in management of type 2
diabetes: a review. J Alternative Compl Med. 2004;10(2):369e378. https://
score of more than nine. Since most of the constituents of PHF
doi.org/10.1089/107555304323062365.
showed in silico SGLT2 inhibitory activity, it can be suggested that 8. Salehi B, Ata A, Anil Kumar NV, et al. Antidiabetic potential of medicinal plants
the glucose-lowering effect of PHF involved the SGLT2 inhibition and their active components. Biomolecules. 2019;9(10):551. https://doi.org/
10.3390/biom9100551.
pathway. Moreover, the individual molecules having high docking
9. Adinortey MB, Agbeko R, Boison D, et al. Phytomedicines used for diabetes
scores can be further studied using higher models. mellitus in Ghana: a systematic search and review of preclinical and clinical
evidence. Evid Based Complement Alternat Med. 2019:6021209. https://doi.org/
5. Conclusion 10.1155/2019/6021209, 2019.
10. Lee J, Noh S, Lim S, Kim B. Plant extracts for type 2 diabetes: from traditional
medicine to modern drug discovery. Antioxidants. 2021;9(10):81. https://
This study concluded that the polyherbal formulation, doi.org/10.3390/antiox10010081.
comprised of 7 herbs viz. A. roxburghiana, C. pareira, S. glabra, D. 11. Semwal DK, Kumar A, Aswal S, Chauhan A, Semwal RB. Protective and thera-
peutic effects of natural products against diabetes mellitus via regenerating
indica, R. cinerea, T. sinensis and C. longa, showed significant pancreatic b-cells and restoring their dysfunction. Phytother Res. 2021;35(3):
glucose-lowering effect via inhibiting SGLT2 protein. The levels of 1218e1229. https://doi.org/10.1002/ptr.6885.
SGOT, SGPT, bilirubin, cholesterol and triglycerides were signifi- 12. Hsia DS, Grove O, Cefalu WT. An update on sodium-glucose co-transporter-2
inhibitors for the treatment of diabetes mellitus. Curr Opin Endocrinol Diabetes
cantly decreased in the PHF-fed rats. The toxicity study, as well as Obes. 2017;24(1):73e79. https://doi.org/10.1097/MED.0000000000000311.
histopathological examination, revealed the nontoxic nature of the 13. Alicic RZ, Neumiller JJ, Johnson EJ, Dieter B, Tuttle KR. Sodium-glucose
formulation. Hence, in view of the antidiabetic effects in animal cotransporter 2 inhibition and diabetic kidney disease. Diabetes. 2019;68(2):
248e257. https://doi.org/10.2337/dbi18-0007.
models, the PHF can be developed as a novel antihyperglycemic 14. Cai T, Ke Q, Fang Y, et al. Sodium-glucose cotransporter 2 inhibition suppresses
agent. However, further studies are needed to consider it for clinical HIF-1a-mediated metabolic switch from lipid oxidation to glycolysis in kidney
use. tubule cells of diabetic mice. Cell Death Dis. 2020;11:390. https://doi.org/
10.1038/s41419-020-2544-7.
15. FDA. Sodium-glucose cotransporter-2 (SGLT2) inhibitors. U.S. Food and drug
Animal ethical approval administration. Contents published on 08/20/2018. https://www.fda.gov/
drugs/postmarket-drug-safety-information-patients-and-providers/sodium-
The animal studies were conducted at M.M. College of Phar- glucose-cotransporter-2-sglt2-inhibitors; 2018.
16. Milder TY, Stocker SL, Day RO, Greenfield JR. Potential safety issues with use of
macy, Ambala with ethical approval No. MMCP/IAEC/55/2019. The sodium-glucose cotransporter 2 inhibitors, particularly in people with type 2
animals were maintained as per Control and Supervision of diabetes and chronic kidney disease. Drug Saf. 2020;43:1211e1221. https://

10
A. Kumar, A.S. Negi, A. Chauhan et al. Journal of Traditional and Complementary Medicine xxx (xxxx) xxx

doi.org/10.1007/s40264-020-01010-6. 297e304. https://doi.org/10.2174/2215083805666190124162640.

17. Kumar A, Aswal S, Semwal RB, Chauhan A, Semwal DK. Insights on the phar- 44. Kumar A, Aswal S, Chauhan A, et al. Antidiabetic effect of aqueous-ethanol
macological, phytochemical and ethnobotanical aspects of Artemisia rox- extract from the aerial parts of Artemisia roxburghiana. Nat Prod Res Online
burghiana: a rather less explored but therapeutically important species of first. 2020. https://doi.org/10.1080/14786419.2020.1858414.
lower Himalayas. Phytochemistry Rev. 2019;18:199e214. https://doi.org/ 45. Tech Virginia. SOP: Retro-Orbital Blood Collection in the Rat (Version 1). Uni-
10.1007/s11101-018-9589-6. versity Veterinarian and Animal Resources; 2017. Published on 12/12/2017.
18. Joshi RK, Satyal P, Setzer WN. Himalayan aromatic medicinal plants: a review Online access from https://ouv.vt.edu/content/dam/ouv_vt_edu/sops/small-
of their ethnopharmacology, volatile phytochemistry, and biological activities. animal-biomedical/sop-rat-blood-collection-retro-orbital.pdf.
Medicine. 2016;3(1):6. https://doi.org/10.3390/medicines3010006. 46. Pariyani R, Ismail IS, Azam AA, Abas F, Shaari K, Sulaiman MR. Phytochemical
19. Kumar A, Aswal S, Semwal RB, Chauhan A, Joshi SK, Semwal DK. Role of plant- screening and acute oral toxicity study of Java tea leaf extracts. BioMed Res Int.
derived alkaloids against diabetes and diabetes-related complications: a 2015;2015:742420. https://doi.org/10.1155/2015/742420.
mechanism-based approach. Phytochemistry Rev. 2019;18(5):1277e1298. 47. Wadaan MA. Effects of mercury exposure on blood chemistry and liver histo-
https://doi.org/10.1007/s11101-019-09648-6. pathology of male rats. J Pharmacol Toxicol. 2009;4(3):126e131. https://
20. Kumari S, Anmol Bhatt V, Suresh PS, Sharma U. Cissampelos pareira L.: a review doi.org/10.3923/jpt.2009.126.131.
of its traditional uses, phytochemistry, and pharmacology. J Ethnopharmacol. 48. Benjamin N, Kushwah A, Sharma RK, Katiyar AK. Histopathological changes in
2021;274:113850. https://doi.org/10.1016/j.jep.2021.113850. liver, kidney and muscles of pesticides exposed malnourished and diabetic rats.
21. Semwal DK, Semwal RB, Vermaak I, Viljoen A. From arrow poison to herbal Int J Exp Biol. 2006;44:228e232.
medicine - the ethnobotanical, phytochemical and pharmacological signifi- 49. Kuntal BK, Aparoy P, Reddanna P. EasyModeller: a graphical interface to
cance of Cissampelos (Menispermaceae). J Ethnopharmacol. 2014;155(2): MODELLER. BMC Res Notes. 2010;3:226. https://doi.org/10.1186/1756-0500-3-
1011e1028. https://doi.org/10.1016/j.jep.2014.06.054. 226.
22. Chopra RN, Chopra IC, Handa KL, Kapur LD. Chopra's Indigenous Drugs of India. 50. Laskowski RA, Jabłon  ska J, Pravda L, Varekova RS, Thornton JM. PDBsum:
second ed. Calcutta: Char UN and Sons, Ltd; 1958:412. structural summaries of PDB entries. Proteome Sci. 2018;27:129e134. https://
23. Kirtikar KR, Basu BD. Indian Medicinal Plants. 2nd ed 1. Allahabad: L.M. Basu; doi.org/10.1002/pro.3289.
2004:94. 51. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V. Global and
24. Semwal DK, Semwal RB. Efficacy and safety of Stephania glabra: an alkaloid- regional mortality from 235 causes of death for 20 age groups in 1990 and
rich traditional medicinal plant. Nat Prod Res. 2015;29(5):396e410. https:// 2010: a systematic analysis for the Global Burden of Disease Study 2010.
doi.org/10.1080/14786419.2014.955487. Lancet. 2012;380(9859):2095e2128.
25. Pullaiah T, Naidu KC. Antidiabetic Plants in India and Herbal Based Antidiabetic 52. Taj M, Mathur Y, Hassan MDI. InstaDock: a single-click graphical user interface
Research. New Delhi: Regency Publications; 2003:168e169. for molecular docking-based virtual high-throughput screening. Brief Bio-
26. Chunekar KC. Bhavprakash Nighantu (Indian Materia Medica), Hindi Edition of inform. 2020. https://doi.org/10.1093/bib/bbaa279. bbaa279.
Bhavaprakasa Nighaṇṭu of  vamisra (1600 A.D.). Varanasi, India: Chau-
Srı Bha 53. Alam F, Saqib QN, Ashraf M. Zanthoxylum armatum DC extracts from fruit, bark
khambha Bharati Academy; 2015. and leaf induce hypolipidemic and hypoglycemic effects in mice-in vivo and
27. Aswal S, Kumar A, Semwal RB, et al. Drimia indica: a plant used in traditional in vitro study. BMC Compl Alternative Med. 2018;18(1):68. https://doi.org/
medicine and its potential for clinical uses. Medicina. 2019;55(6):255. https:// 10.1186/s12906-018-2138-4.
doi.org/10.3390/medicina55060255. 54. Panda A, Jena S, Sahu PK, Nayak S, Padhi P. Effect of polyherbal mixtures on the
28. Dobhal MP, Joshi BC. Chemical investigations of Roylea elegans Wall Part I. treatment of diabetes. ISRN Endocrinol. 2013:934797. https://doi.org/10.1155/
Herba Pol. 1979;25:95e97. 2013/934797, 2013.
29. Bhatt UP, Sati SC, Bahuguna RP, Semwal RB, Semwal DK. Two antidiabetic 55. Choudhury H, Pandey M, Hua CK, et al. An update on natural compounds in the
constituents from Roylea cinerea (D.Don) Baill. Nat Prod Res. 2018;32(11): remedy of diabetes mellitus: a systematic review. J Tradit Complement Med.
1281e1286. https://doi.org/10.1080/14786419.2017.1331229. 2017;8(3):361e376. https://doi.org/10.1016/j.jtcme.2017.08.012.
30. Bhatia A, Buttar HS, Arora R, et al. Antiproliferative effects of Roylea cinerea (D. 56. Petchi RR, Vijaya C, Parasuraman S. Antidiabetic activity of polyherbal formu-
Don) Baillon leaves in immortalized L6 rat skeletal muscle cell line: role of lation in streptozotocin - nicotinamide induced diabetic wistar rats. J Tradit
reactive oxygen species mediated pathway. Front Pharmacol. 2020;11:322. Complement Med. 2014;4(2):108e117. https://doi.org/10.4103/2225-
https://doi.org/10.3389/fphar.2020.00322. 4110.126174.
31. Hegde S, Jayaraj M. A review of the medicinal properties, phytochemical and 57. Singhal S, Rathore AS, Lohar V, Dave R, Dave J. Pharmacological evaluation of
biological active compounds of tinospora sinensis (lour.) Merr. J Biol Act Prod "sugar remedy," a polyherbal formulation, on streptozotocin-induced diabetic
Nat. 2016;6(2):84e94. https://doi.org/10.1080/22311866.2016.1185968. mellitus in rats. J Tradit Complement Med. 2014;4(3):189e195. https://doi.org/
32. Ahmad S, Zahiruddin S, Parveen B, et al. Indian medicinal plants and formu- 10.4103/2225-4110.127800.
lations and their potential against COVID-19-preclinical and clinical research. 58. Duraiswamy A, Shanmugasundaram D, Sasikumar CS, Cherian SM, Cherian KM.
Front Pharmacol. 2021;11:2470. https://doi.org/10.3389/fphar.2020.578970. Development of an antidiabetic formulation (ADJ6) and its inhibitory activity
33. Saha S, Ghosh S. Tinospora cordifolia: one plant, many roles. Ancient Sci Life. against a-amylase and a-glucosidase. J Tradit Complement Med. 2016;6(3):
2012;31(4):151e159. https://doi.org/10.4103/0257-7941.107344. 204e208. https://doi.org/10.1016/j.jtcme.2014.12.006.
34. Zhang DW, Fu M, Gao SH, Liu JL. Curcumin and diabetes: a systematic review. 59. Porwal M, Khan NA, Maheshwari KK. Evaluation of acute and subacute oral
Evid Based Complement Alternat Med. 2013:636053. https://doi.org/10.1155/ toxicity induced by ethanolic extract of Marsdenia tenacissima leaves in
2013/636053, 2013. experimental rats. Sci Pharm. 2017;85:29. https://doi.org/10.3390/
35. Kim S, Ko S, Kim Y, et al. Determination of Curcuma longa L. (Turmeric) leaf scipharm85030029.
extraction conditions using response surface methodology to optimize 60. Szkudelski T. Streptozotocin-nicotinamide-induced diabetes in the rat. Char-
extraction yield and antioxidant content. J Food Qual. 2019:7575206. https:// acteristics of the experimental model. Exp Biol Med. 2012;237(5):481e490.
doi.org/10.1155/2019/7575206, 2019. https://doi.org/10.1258/ebm.2012.011372.
36. Ahn DR, Lee EB, Kim BJ. Antioxidant and lifespan extending property of 61. Chawla A, Chawla R, Jaggi S. Microvasular and macrovascular complications in
quercetin-3-odirhamnoside from Curcuma longa L. In Caenorhabditis elegans. diabetes mellitus: distinct or continuum? Indian J Endocrinol Metabol.
J Korean Soc Appl Biol Chem. 2014;57(6):709e714. https://doi.org/10.1007/ 2016;20(4):546e551. https://doi.org/10.4103/2230-8210.183480.
s13765-014-4200-3. 62. Kannel WB. Lipids, diabetes, and coronary heart disease: insights from the
37. Joshi P, Joshi S, Semwal DK, Bisht A, Sharma S, Dwivedi J. Chemical composi- Framingham Study. Am Heart J. 1985;110(5):1100e7. https://doi.org/10.1016/
tion, antioxidative and antimicrobial activities of turmeric spent oleoresin. Ind 0002-8703(85)90224-8.
Crop Prod. 2021;162:113278. https://doi.org/10.1016/j.indcrop.2021.113278. 63. Midha T, Krishna V, Shukla R, et al. Correlation between hypertension and
38. Parasuraman S, Thing GS, Dhanaraj SA. Polyherbal formulation: concept of hyperglycemia among young adults in India. World J Clin Cases. 2015;3(2):
ayurveda. Phcog Rev. 2014;8(16):73. https://doi.org/10.4103/0973- 171e179. https://doi.org/10.12998/wjcc.v3.i2.171.
7847.134229. 64. Petrie JR, Guzik TJ, Touyz RM. Diabetes, hypertension, and cardiovascular dis-
39. Chhetri DR, Parajuli P, Subba GC. Antidiabetic plants used by Sikkim and ease: clinical insights and vascular mechanisms. Can J Cardiol. 2018;34(5):
Darjeeling Himalayan tribes, India. J Ethnopharmacol. 2005;99(2):199e202. 575e584. https://doi.org/10.1016/j.cjca.2017.12.005.
https://doi.org/10.1016/j.jep.2005.01.058. 65. Bari MW, Islam MM, Khatun M, et al. Antidiabetic effect of Wedelia chinensis
40. Modak M, Dixit P, Londhe J, Ghaskadbi S, Devasagayam TP. Indian herbs and leaf extract in alloxan induced Swiss albino diabetic mice. Clin Phytosci. 2020;6:
herbal drugs used for the treatment of diabetes. J Clin Biochem Nutr. 58. https://doi.org/10.1186/s40816-020-00197-6.
2007;40(3):163e173. https://doi.org/10.3164/jcbn.40.163. 66. Tsushima Y, Lansang MC, Makin V. The role of SGLT-2 inhibitors in managing
41. Lale SK, Chandra R, Rath C, Mangal A, Murthy SN. Exploration of medicinal type 2 diabetes. Cleve Clin J Med. 2020;88(1):47e58. https://doi.org/10.3949/
plants of hoshangabad forest division (territorial), Madhya Pradesh, with ccjm.88a.20088.
special reference to ayurveda. J Drug Res Ayurvedic Sci. 2019;4(4):175e184. 67. Wang JY, Cheng YZ, Yang SL, et al. Dapagliflozin attenuates hyperglycemia
https://doi.org/10.5005/jdras-10059-0085. related osteoporosis in ZDF rats by alleviating hypercalciuria. Front Endocrinol.
42. OECD. Acute oral toxicity - acute toxic class method. OECD Guidelines for 2019;10:700. https://doi.org/10.3389/fendo.2019.00700.
testing of chemicals. https://doi.org/10.1787/20745788; 2002. 68. Kaneria M, Parmar J, Rakholiya K. Molecular docking and drug design of phy-
43. Semwal DK, Dahiya RS, Joshi N, et al. Preclinical and clinical studies to evaluate toconstituents from Couroupita guianensis-An in silico perspective.
the effect of Carica papaya leaf extract on platelets. Curr Tradit Med. 2018;4: J Pharmacogn Phytochem. 2019;8(6):53e60.

11