Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

https://doi.org/10.1007/s40011-023-01499-8

RESEARCH ARTICLE

Panchvalkal (an Ayurvedic Formulation) Proliferates Pancreatic

β‑Cells and Increases the Potency of Conventional drug
Glibenclamide
Ruchita Tripathi1 · Amit Ranjan2 · Raghvendra Pratap3 · Anil Kumar Singh1 · Rajesh Kumar Singh1

Received: 31 March 2023 / Revised: 8 May 2023 / Accepted: 9 May 2023

© The Author(s), under exclusive licence to The National Academy of Sciences, India 2023

Abstract Diabetes is a common metabolic syndrome in rats as used as in-vivo model with Streptozotocin diabetes
Indian population. The drugs used for its management based induction. The results show that the integration of the Ayur-
on enzyme and hormone. The development of resistance vedic formulation is safe and potentiating the efficacy of
against conventional drugs in diabetic patients is a major Glibenclamide.
challenge for clinicians to treat the patients effectively.
Panchavalkala is a polyherbal formulation, described in Keywords Panchavalkala · Antidiabetic · Polyherbal
Ayurveda for management of many clinical ailments and Ayurvedic formulation · MIN-6 cell line · Waster rats ·
potentiating the antibacterial drugs in recent studies. In this Streptozotocin · Histopathology
study it has been evaluated for integration with conventional
drug, Glibenclamide for potentiating the efficacy and safety
for management of patient with diabetes mellitus in north Introduction
Indian population. The methodology of Panchvalkala kwath
preparation is according to protocol described in Ayurvedic The incident rate of metabolic disorder is exponentially
Pharmacopoiea of India (API) and evaluated for safety and raised worldwide and is becoming a major health trouble in
efficacy alone and integrated with conventional drug, Glib- upcoming periods, Diabetes is one of the global epidemic
enclamide in in vitro as well as in-vivo model. The waster challenge and fourth leading cause of mortality in develop-
ing countries [1]. Global diabetes prevalence is estimated to
be 9.3% of world population were affected with Diabetes in
Significant Statement Panchvalkala is an Ayurvedic formulation 2019 and is expected to 10.9% by 2045 [2]. Approximately
which is described in ancient literature for treatment of various 90–95% diabetic person struggle with Diabetes Mellitus.
clinical ailments. Although, there is no literature described it as
an antidiabetic drug. This study revealed the clinical importance
Diabetes is represented by hyperglycemia, which can directly
of the formulation either alone or integrated with conventional associated with many metabolic complications like, retinop-
drug. More surprising it is not only helped in managing diabetic athy, nephropathy and neuropathy, cardiovascular disorders,
condition but also improve quality of life and proven that it etc. [3, 4]. Hyperglycemia can be controlled by appropriate
proliferate the beta-cells of pancreas.

1
* Rajesh Kumar Singh Department of Dravyaguna, Faculty of Ayurveda,
rkszoology@gmail.com Institute of Medical Sciences, Banaras Hindu University,
Varanasi 221005, India
Ruchita Tripathi
2
tripathi.ruchita85@gmail.com Department of Kayachikitsa, Institute of Medical Sciences,
Banaras Hindu University, Varanasi 221005, India
Amit Ranjan
3
amitranajn1111@gmail.com Department of Peditrics, Institute of Medical Sciences,
Banaras Hindu University, Varanasi 221005, India
Raghvendra Pratap
raghvendrapratap87@gmail.com
Anil Kumar Singh
anilkumar.singh113@gmail.com

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 R. Tripathi et al.

management of altered regulatory enzymes responsible for to evaluate the integrated effect of conventional drug, Glib-
balancing glucose homeostasis. Beta cell of Pancreatic islets enclamide with Panchavalkal kwatha in Diabetes mellitus
produce a peptide harmone called insulin which play crucial patients for management of diabetes mellitus in north Indian
role for glucose metabolism [5]. The major complication population.
in diabetes represented by hyperglycemia due to absolute
or relative insulin deficiency in both 1 and 2 type of diabe-
tes [6, 7]. In 1 type of diabetes, beta cells of pancrease are Material and Methods
destroyed by some immunological cells of immune system,
like macrophages, different type of cytokines/T lymphocyte Collection and Identification of Plant Materials
triggered via autoimmunity. Besides this in Type 2 diabetes
are the outcome of comparative insulin deficiency coupled The plant materials Vata (F. bengaenesis Linn.), Udumbara
with insulin resistance and that are unable to compensate for (F. glomerata Roxb.), Ashvatha (F. religiosa Linn.) and
the insulin resistance. Beta cells of pancreatic are damage or Plaksha (F. lacor Buch-Ham.) were collected locally (Vara-
become inactive due to consistently hyperglycemic/hyperli- nasi). Parisha (T. populanea Soland ex correa) was collected
pidimic condition, many immune-inflammatory mediators from rural area of maharashtra. These plant materials were
like resistin, TNF-α and IL-6 that promote insulin-resist- collected during December 2016 to January 2017. All the
ance. Adipocytokines released from the adipose tissue play plants ware identified by Prof. K. N. Dwivedi and Prof. Anil
a crucial role for maintaining glycemic homeostasis. In Kumar Singh, Department of Dravyaguna, Faculty of Ayur-
this way, beta cell of pancrease role may be a strategically veda, IMS, BHU and voucher were deposited with following
move towards for avoidance and management of diabetes. accession number:
Many research has been proceed for searching novel and
economically affordable antidiabetic natural drug with fewer 1. Vata (Ficus bengalenesis Linn.) :DG/18/174
or noside and improve the function of pancreatic beta cell or 2. Ashvatha (Ficus religiosa Linn) : DG/18/175
able to increase the cell density of beta cell. Generally natu- 3. Udumbara (Ficus glomerata Roxb.) : DG/18/176
ral compounds are a secondary metabolites isolated from 4. Plaksha (Ficus lacor Buch-Ham.) : DG/18/177
different plants, animals, and microorganisms [8]. Natural 5. Parisha (Thespesia populanea Soland ex correa) :DG/18/178
metaboliters participate a chief role in the finding of novel
bioactive compound having lot of therapeutic properties like
hypoglycemic, hypolipidemic and free radical scavengering Preparation of Decoction
activity [5]. Huge number of herbal drug described with
hypoglycemic properties. Over 400 plants either in single Coarse powder (yavkut) of Panchavalkala were prepared
form or in formulation is used as an antidiabetic drug. The from Ayurvedic pharmacy, Faculty of Ayurveda, Institute of
bioactive compounds of these drugs showed scientificant Medical Science, BHU Varanasi. 50 g of Yavkut was soaked
evaluation for management of Diabetes Mellitus. Panchav- in four times its weight in water for the whole night. The
alkala (An Ayurvedic polyherbal formulation) is an asso- next day, it was heated while being constantly stirred until it
ciation of five adstringent plants namely, Ashvatha(Ficus had dwindled to one-fourth of its initial volume. The liquid
religiosa Linn.), Vata (Ficus bengalenesis Linn.), Udumbara was filtered through four folded clean cotton cloth and the
(Ficus glomerata Roxb.), Plaksha (Ficus lacor Buch-Ham.), filtrate was collected [8].
Parisha (Thespesia populanea Solandexcorrea), which
exhibit different anti-inflammatory, antioxidant, wound
healing, antidiabetic, antibiotic, immunomodulatory, and In Vitro Study
adstringent properties. All five drug of Panchvalkal is kap-
hapittasamaka due to its kasaya rasa, ruksaguna, sitavirya MIN-6 cells were procured from National Centre for Cell
and katu Dosakarma, the drug seems to be rationale for man- Sciences (NCCS) Pune, Department of biotechnology,
agement of Madhumeha [9] Government of India for cell culture. The cells were grown
Recently lot of studies have been reported the develop- in DMEM with high-glucose, sodium bicarbonate, L-glu-
ment of resistance against conventional drugs in diabetic tamine, and sodium pyruvate, which was supplemented with
patients which is challenge for clinicians to treat the patients 10% foetal bovine serum (FBS) and 1% antibiotics-antimy-
effectively. Several researches in integrative medicine show cotic solution (10,000 units/mL of penicillin G, 10 mg/mL
that the integration of conventional drugs with polyherbal of streptomycin sulphate, and 25 g/mL amphotericin B in
formulation improves the efficiency of the conventional 0.9% normal saline). Trypsinization was used to extract
drugs and reduces their side effects, leading to better life of around 70–80% of confluent cells at the log phase (1X
the patients. Therefore, the aim and objective of this work is Trypsin–EDTA Solution, Himedia) [10].

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Panchvalkal (an Ayurvedic Formulation) Proliferates Pancreatic β‑Cells and Increases the…

Cell Toxicity and Proliferation Study Diabetic Model Development

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetra- Streptozotocin (STZ) was dissolved in 0.1 M cold citrate
zolium bromide) solution, which is cleaved by the mito- buffer (pH 4.5, freshly prepared) and intra peritoneally
chondrial enzyme succinate dehydrogenase enzyme in administered at a dose of 60 mg/kg after overnight fasted
living cells into water-insoluble violet coloured formazan rats. The rats were received dextrose solution (5%) after
crystals, was used for the cell toxicity and proliferation 6 h of the injection for next 24 h to avoid STZ induced
experiments. The process was carried out as our study death. Diabetic condition was confirmed after 72 h of
team had previously described. The percentage cell viabil- induction through tail vein blood glucose levels measur-
ity was calculated to compare with test and standard, then ing using glucose meter using strips. These diabetic rats
­IC50 was consider for the same [11, 12]. were kept for 14 days under normal laboratory condition
for examine the blood glucose levels. The blood glucose
was again examined and animals with more than 250 mg/
dl blood glucose level were selected for the study [14].
In‑vivo Study

Ethics and Experimental Animal Antidiabetic Evaluation

In vivo studies were carried out after approval with the The rats were divided into five groups with six rats in
institutional ethical committee (Dean/2018/EC/331) of the each group. Group 1: normal control rats received distiled
Institute of Medical Sciences, BHU, Varanasi, India. The water; group 2: STZ-induced diabetic rats were treated
animals (waster rats of either sex weighing 150–180 g of with glibenclamide (0.517 mg/kg b.wt); group 3: STZ-
age from 6 to 8 weeks old) were procured from the cen- induced diabetic rats were treated with Panchavalkala kwa-
tral animal house, Institute of Medical Sciences, Banaras tha; group 4: STZ-induced diabetic rats were treated with
Hindu University (BHU), Varanasi, India. The animal Panchavalkala kwatha + glibenclamide; group 5: Normal
were kept in polypropylene cages with free supply of water rats. The distil water, Panchavalkala kwatha and gliben-
and a standard pellet diet (Agro Corporation Pvt. Ltd., clamide were administered orally to its respective group
Bengaluru, India) following CPCSEA guidelines for the animals for 28 days. The fasting blood glucose level and
experimental study. body weight were estimated every week (0, 7, 14, 21, and
28 day). At the end of the experimental period, the ani-
mals fasted overnight and blood was collected for various
Oral Acute Toxicity Assessment biochemical estimations. The animals were sacrificed by
cervical dislocation. Organs like the liver, pancreas and
According to the Organization for Economic Cooperation skeletal muscle were dissected out, immediately rinsed in
and Development (OECD test 425) defined technique, the ice cold saline and stored for further biochemical estima-
oral acute toxicity was evaluated. Briefly, animals were tions [15].
maintained on fasting the day before an experiment. The
next day, rats in experimental groups 1 and 2 received a
single dosage of Panchavalkalakwatha at 2000 mg/kg and Dose Calculation
5000 mg/kg body weight, respectively, whereas rats in the
control group received distilled water. To gather signs and The dose of Panchavalkala kwatha for adult is 50 g/day. The
symptoms of toxicity, all animals were inspected individu- dose for experimental animals (rat) was calculated by extrap-
ally after the first 30 min after dosage, with specific care olating the human dose to animals (10.33 ml/kg body weight
given during the first 4 h, and monitoring continued from mg/kg) based on the body surface area ratio by referring to
24 h up to the 14th day [12, 13]. Investigating the possibil- the standard table of Paget and Barnes [8].This was admin-
ity of tremors, convulsions, salivation, diarrhoea, lethargy, istered to animals orally with the help of a gastric catheter
and sleepiness was done. One of the toxicity measures was sleeved to syringe. The drugs were administered twice a day.
the live weight of the animals, which was checked on days
0, 7, and 14 after dosage. The animals were killed via cervi-
cal dislocation of sedated animals at the conclusion of the Estimation of Biochemical Parameters
experiment, and the organs, including the brain, stomach,
liver, spleen, lungs, kidneys, muscles, oesophagus, and small The serum lipid profiles and vital organ function tests were
intestine, were taken to assess histological alterations [11]. analysed using commercially available kits. All above

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 R. Tripathi et al.

biochemical parameters were estimated using semi-auto Pancreatic Beta Cells Proliferation Potential in MIN‑6
analyser [16]. Cell Lines

Statistical Analysis MIN-6 cell lines treated with Panchavalkala Kwatha

(0.78 µg/ml) and showed significantly increment in pancre-
All the data of this study, expressed as mean ± SEM which atic beta cell proliferation rate with time. The cell (MIN-6
were analysed by one-way analysis of variance (ANOVA), pancreatic beta cell) treated with Panchavalkala kwatha
followed by Dunnett’s test for multiple comparisons. The showed higher cell proliferation as compare to control
prism GraphPad version 5.0 software was used where (Fig. 2).
P < 0.05 was considered as statistically significant.
Study of Acute Toxicity
Histology
The toxic effect of Panchavalkala kwath was examined by
For histological study tissues were obtained from animals oral acute toxicity study followed by standard protocol men-
treated with Panchavalkala kwatha, control animal and pro- tion in OECD guide line (test number 425). After that vital
cessed for further examination [17]. Solution of 4% neutral organs tissue were used for examine the alteration in cellular
saline formalin was used for fixation of excised tissues, after architecture due to toxicity. In experimental rats, administra-
that it allow to undergo dehydrated in alcohol and then fixed tion of Panchavalkala kwatha extract orally at 2000 mg/kg
permanently in paraffin wax. The Microtome (Leica Biosys- and 5000 dose not induced any signs and symptom due to
tems, Germany) was used for section cutting(5 μm) followed toxicity and up to 14 days no any rats were died. The Pan-
by staining with haematoxylin and eosin. light microscope chavalkala kwatha extract was found no any kind of toxicity
(Dwinter) were used for capturing the images of all tissue in rats up to 5000 mg/kg of body weight at very high oral
sections.

Results

Cell Cytotoxicity Assay

In vitro action of Panchavalkala kwatha using Pancreatic

β-Cell lines (MIN6 Cell line). To evaluating even if Pan-
chavalkala kwatha showed any cytotoxic activity, MTT assay
was execute. After using the panchavalkala extracts at con-
centrations ranging from 0.78 to 400 µg/ml, the viability
retained up to 100% (p < 0.001), still at very high concen- Fig. 2  Pancreatic beta cell proliferation potential of Panchavalkala
tration (400 µg/ml) (Fig. 1). This evaluation reported that Kwatha(0.78µG/ml) in MIN-6 cells. Proliferation potency of beta
cell treated with Panchavalkala Kwatha in MIN-6 cells line. Obtained
Panchavalkala Kwatha not show any kind of celltoxicity. results are normalized to 100 based on control readings. Values are
means ± SD

Fig. 1  Effects of Panchavalkala

Kwatha on viability of β-cells.
MTT was execute to analyse
the cytotoxicity of Panchaval-
kala Kwatha. For cell viability
examination Panchavalkala
Kwatha treated MIN6 cells with
low to high concentrations (Ctrl
N = Control Negative; normal
rat without any treatment; Ctrl
P = Control Positive, Strepto-
zotocin induced diabetic rat
without any treatment)

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Panchvalkal (an Ayurvedic Formulation) Proliferates Pancreatic β‑Cells and Increases the…

Histological Study (H & E Staining)

Effect of Panchavalkala Kwatha on Level of Glucose

in Blood and Body Weight

After administration of STZ significant increment

(P < 0.001) were reported in blood glucose levels com-
pared to the normal control rats. After oral treatment
of combination of Panchavalkala kwatha (10.33 ml/kg
Fig. 3  Acute toxic showed the effect of Panchavalkala kwatha on b. wt) and glibenclamide (0.517 mg/kg body weight)
the body weight of rats treated with very high dose (2000 mg/kg and
5000 mg/kg body weight) in 14 day treatment the increased level of sugar decreased more remarkably
(P < 0.001). Glibenclamide and Panchavalkala kwatha
individually also showed the significant decline in blood
dose. Meanwhile, body weight of treated model not show glucose level when compared to diabetic rats to diabetic
any altered result during the whole acute toxic study and at control rats (Table 1). After 28 days utmost reduction was
the end of study the rats showed normalized body weight detected. STZ administration reduce the body weight sig-
(Fig. 3). After 14 th day rat were scarified for histological nificantly (P < 0.001) in diabetic rats in contrast to normal
examination of vital organ.No any significant changes has control rats. When diabetic rats, treated with of Panchav-
been reported at the level of tissue or cellular architecture alkala kwatha and glibenclamide in combined form body
in treated rats. The histological slides conformed the oral weight significantly improve (P < 0.01 and P < 0.001) as
administration of Panchavalkala kwatha. (Fig. 4). compared to diabetic control rats (Fig. 5).

Fig. 4  Evaluation of histology

of toxic effect of Panchavalkala
formulation in different vital
organ. HE staining was used
for visualization of the cellular
architecture of tissue of differ-
ent organ of treated group and
control group

Table 1  Chronic effects of Panchavalkala kwatha on fasting blood glucose levels in control group and different experimental groups
Treatment Dose (ml/kg) 14 day of STZ injection 1st week 2nd week 3rd week 4rd week

Normal control water 63.06 ± 17.22 68.38 ± 21.22 70.78 ± 17.26 71.72 ± 18.56 72.01 ± 10.33
Diabetic control water 387 ± 19.13 380.64 ± 20.12 390.8 ± 22.02 384 ± 22.78 390.2 ± 21.65
Panchavalkala 10.33 ml/kg b.w 372.5 ± 16.44 290 ± 14.11 173.44 ± 14.26 143 ± 16.64 130.22 ± 13.35
Panchaval- 10.33 ml/kg 385.83 ± 20.95 210 ± 16.52 135.56 ± 20.49 115.21 ± 11.47 113.12 ± 11.85
kala + Gliben- b.w + 0.517 mg/
clamide kg b.wt
Glibenclamibe 0.517 mg/kg b.wt 407 ± 17.22 266.3 ± 15.32 152.92 ± 21.27 99.83 ± 18.56 85.57 ± 17.55

13
 R. Tripathi et al.

Fig. 5  In STZ induced diabetic

rats effect of Panchaval-
kala kwatha on blood sugar
levels, all value is expressed
as mean ± S.E.M. (n = 6).
# = P < 0.001 in contrast to
analogous values of the normal
control.* = . P < 0.001 in con-
trast to analogous values of the
diabetic control

Table 2  Effect of Treatment Dose(ml/kg) SGOT(U/L) SGPT(U/L) Creatinine(mg/dl)

Panchavalkalakwatha on
some liver and kidney function Normal control Water 147.04 ± 6.65 91.35 ± 2.54 050 ± 0.03
markers of diabetic rats
Diabetic control Water 246.57 ± 13.42 163.04 ± 3.63 0.76 ± 0.02
(n = 6 ± SE)
Panchavalkala 10.33 ml/kg b.w 174.85 ± 3.97 127.13 ± 4.54 0.59 ± 0.04
Panchaval- 10.33 ml/kg 158.62 ± 3.35 105.29 ± 4.50 0.55 ± 0.03
kala + Glibencla- b.w + 0.517 mg/kg
mide b.wt
Glibenclamibe 0.517 mg/kg b.wt 150.25 ± 4.6 91.1 ± 5.33 0.53 ± 0.02

Effect of Panchavalkalakwatha on Creatinine, SGOT,

SGPT

Table 2 shows, the effect of Panchavalkalakwatha on serum

SGPT, serum SGOT, creatinine in group of diabetic rats.
The diabetic rats induced by STZ showed significantly
altered level of different liver and kidney function biochem-
ical marker like SGOT, SGPT, creatinine in comparison
with normal control rats. After oral administration of Pan-
chavalkala kwatha and glibenclamide significant reduction
(P < 0.001, P < 0.01 and P < 0.05) has been reported in cre- Fig. 6  Effect of Panchavalkala kwatha on weight of body in dia-
betic rats induced by STZ. All data are expressed as mean ± SEM
atinine SGOT and SGPT levels in diabetic rats compared to (n = 6). #P < 0.001 diabetic control, Panchavalkala kwath(10.33 ml/
diabetic control rats. (Figs. 5, 6, 7.) kg b.w), comparision of normal with Panchavalkala kwath(10.33 ml/
kg b.w) + Glibenclamide(0.517 mg/kg b.wt);*P < 0.05 Pan-
. chavalkala kwath(10.33 ml/kg b.w) compared with diabetic
control;**P < 0.01Panchavalkala kwath(10.33 ml/kgb.w) + Glibencla-
mide(0.517 mg/kg compared with diabetic control; ***P < 0.001 Pan-
Effect of Panchavalkalakwatha on Lipid Profiles chavalkala kwath(10.33 ml/kg b.w), Panchavalkala kwath(10.33 ml/
kg b.w) + Glibenclamide(0.517 mg/kg b.wt) compared with diabetic
As compared to normal control rats the STZ-induced dia- control
betic rats showed significantly (P < 0.001) increased level
of TC, TG, LDL, and VLDL whereas decreased level of b. wt.) and both glibenclamide and Panchavalkala kwatha,
HDL in Table 3. total cholesterol level of group showed very high significant
As compared to normal control rats, diabetic rats induced reduction following the total triglyceride as compare to con-
by STZ showed significantly increased (P < 0.001) level of trol group. The treated group with Panchavalkala kwatha
TC, TG, LDL, and VLDL whereas decreased level of HDL. also show significant reduction but the percentage of reduc-
After 28 days of treatment with glibenclamide (0.517 mg/kg tion is less as compare to above group. The diabetic rats

13
Panchvalkal (an Ayurvedic Formulation) Proliferates Pancreatic β‑Cells and Increases the…

kwatha and glibenclamide both compare to diabetic control

rats. The group treated with Panchavalkala kwatha also show
significant increased level but the percentage of increased
level is less as compare to group treated with both modern
as well as Ayurvedic formulation.

Histological Evaluation of Pancrease

The pancreatic islet cells in the normal control rats showed

the typical cellular population and acini (Fig. 9c). Never-
theless, diabetic control rats showed a tiny and diminished
number of islet cells (Fig. 9a) Especially in the core–cell
Fig. 7  Effect of Panchavalkala kwatha and glibenclamide on liver zone, pancreatic sections from diabetic rats treated with
function markers in diabetic rats induced by STZ. Data represented Panchavalkalakwatha and glibenclamide demonstrated
as mean ± SEM (n = 6). Statistically significant differences were
observed between Normal to Diabetic control: #p < 0.001 and Dia- the regeneration of islet cells’ normal size and cell count
betic control to treated groups with drug: *p < 0.001 (Fig. 9b). The Panchavalkala kwatha-treated normal group
showed no discernible improvement.

induced by STZ show increased level of serum LDL choles-

terol.The above graph show effect of Panchavalkala kwatha Discussion
on elevated level of LDL cholesterol on diabetic rats. After
28 days the group treated with both Panchavalkala kwatha Despite the creation of newer treatments to cure or prevent
and glibenclamide and individually with glibenclamide the condition, diabetes has persisted despite the frequency of
show highly significant reduction in LDL level. The group numerous discoveries. Long-lasting abnormalities and organ
treated with Panchavalkala kwatha also show significant destruction are brought on by prolonged hyperglycemia, par-
reduction similarly. The VLDL cholesterol also increased ticularly in tissues that depend on insulin for glucose uptake.
in diabetic rats induced by STZ. It has been reported that Because of the negative side effects of synthetic medications,
in diabetic rats the Panchavalkala kwatha also reduce the managing diabetes is a very difficult undertaking. Several
level of VLDL cholesterol. After 28 days group treated with herbal plants have been traditionally used for treating diabe-
both Panchavalkala kwatha and glibenclamide and individu- tes mellitus over the years. Due to the presence of flavonoids,
ally with glibenclamide show highly significant reduction phenol, triterpenes, tannins, carbohydrates, and saponins, all
in VLDL level. The % of reduction is high as compare to plants used in the formulation are known to demonstrate
Ayurvedic formulation. After 28 days of treated with gliben- hypoglycemic, hypolipidemic, and antioxidant action. The
clamide and both glibenclamide and Panchavalkala kwatha, treatment of diabetes using the bark of Panchavalkala com-
the group showed highly significant reduction in total cho- ponent plants has been documented in earlier literature [18].
lesterol level as compare to control group. The group treated Their therapeutic efficacy has, however, received only spo-
with Panchavalkala kwatha also show significant reduction radic scientific support. The current study was conducted to
but the percentage of reduction is less as compare to above investigate Panchavalkala kwatha’s anti-diabetic effects in
group (Fig. 8). diabetic-induced albino Wister rats. This study had divided
In the same way, oral administration of glibenclamide in to two categories, i.e. in vivo and in vitro. The MIN 6 cell
(0.517 mg/kg b. wt.) showed highly significant rised level line was used for evaluation the cytotoxic effect of Panchav-
of HDL was obtained in group treated with Panchavalkala alkal kwatha in in vitro study. At very low concentrations

Table 3  Effect of Treatment Dose(ml/kg) TC TG HDL LDL VLDL

Panchavalkala kwatha
administrated orally on serum Normal control Water 94.2 ± 9.4 101.38 ± 12.21 38.75 ± 5.2 32.89 ± 2.2 20.79 ± 1.7
lipid profile (n = 6, mean ± SE)
Diabetic control Water 144 ± 11.7 144.18 ± 11.59 33.09 ± 3.2 77.51 ± 5.2 31.26 ± 2.3
Panchavalkala 10.33 ml/kg b.w 121.34 ± 1.8 127.6 ± 4.4 39.97 ± 2.1 55.33 ± 3.1 28.66 ± 1.6
Panchaval- 10.33 ml/kg 114.79 ± 2.5 124.54 ± 4.7 43.41 ± 1.7 48.41 ± 2.1 25.47 ± 1.3
kala + Gliben- b.w + 0.517 mg/
clamide kg b.wt
Glibenclamibe 0.517 mg/kg b.wt 114 ± 7.9 123.55 ± 5.3 47.64 ± 3.9 42.99 ± 4.1 23.49 ± 1.9

13
 R. Tripathi et al.

Fig. 8  Serum lipid profile study in diabetic rats induced by STZ were observed between Normal to Diabetic control: #p < 0.001 and
treated with Panchavalkala kwatha and glibenclamide. Data repre- Diabetic control to treated groups with drug: *p < 0.001
sented as mean ± SEM (n = 6). Statistically significant differences

to high concentration from 0.78 to 400 µg/mL, the viability it exhibited significant cell proliferation rate. In this way
retained up to 100% (p < 0.001), even at maximum concen- significant increment in proliferation of beta cell was recog-
tration (400 µg/ml). This study verified the nontoxicity of nized in MIN-6 cells after Panchavalkala kwatha treatment.
Panchavalkala kwatha. In cell proliferation study when MIN This increment in rate of proliferation of pancreatic beta cell
6 cell line treated with Panchavalkala kwatha (0.78 µg/ml), of test material symbolize a tremendously helpful criteria

13
Panchvalkal (an Ayurvedic Formulation) Proliferates Pancreatic β‑Cells and Increases the…

Fig. 9  Histopathological section of pancreas of rats. a Diabetic rats minimal (arrows). c Normal rats with normal acini and normal cel-
with minute and reduced number of islet cells. b Diabetic rat treated lular population showing exocrine area, mononuclear cell infiltration
with Panchavalkala kwatha formulation (10.33 mg/kg body weight (arrows)
and glibenclamide 4.35 µg/kg body weight showing vacuolar change

to investigate the hypoglycemic activity, which also prevent level exceeds the renal threshold. The body weight of the
the pancreatic beta cells from destruction caused by gluco- diabetic rats in the current study significantly decreased. By
lipotoxicity during type 2 diabetes or defend from autoim- administering a pure fraction to diabetic rats as a model,
mune mediated damage as in type 1 diabetes. We noticed the the aforementioned condition has been improved. Effec-
preventive action of Panchavalkala kwatha against toxicity in tiveness of kwatha in preventing weight loss, which may
pancreatic cells caused by streptozotocin. Due to our finding, be caused by its capacity to increase plasma insulin levels,
we strongly consider the drug proliferation property contrib- improving hyperglycemic control mechanisms [23]. Dia-
ute significantly to its antidiabetic ability. betes is brought on by the pancreatic beta-cells’ inability
In vivo study, diabetic rat model prepared by induction of to regulate the plasma insulin, which causes an increase in
STZ which is a nitrosourea complex obtained from Strepto- glucose concentration. In reaction to elevated blood sugar
myces achromogenes which induces DNA strand breakage levels, insulin is released. It balances the liver’s production
causing discerning obliteration of pancreatic β-cells, is less and absorption of glucose with the uptake of glucose by
toxic than alloxan [19], and also allows a consistent main- skeletal muscle and adipose tissue. In the study that follows,
tenance of diabetes. The induced diabetic rat treated with glibenclamide-treated diabetic rats show a rise in plasma
Panchavalkal kwath. Oral treatment of both Panchavalkala insulin levels compared to the diabetic control rats. The ele-
kwatha (10.33 ml/kg b.wt) combination with glibenclamide vated decrease in FBS level in rats treated with both kwatha
(0.517 mg/kg b.wt) and Panchavalkalakwath alone reduce and modern medication as compared to modern medication
of glucose level 75% and 64.98%, respectively, against dia- alone suggests that the Panchavalkalakwatha formulation
betic rats. Aqueous extract of bark of one ingradient plant may cause the release of insulin from the pancreatic cells
F. religiosa showed the hypoglycemic activity by promoting that have already been present and may restore those that
the pancreatic secreting of insulin by exixting beta cell of STZ has destroyed. An ideal anti-diabetic medicine prevents
islets [18]. One active compound leucopelargonin has been hypoglycemia while reducing hyperglycemia and enhancing
reported in extract of F. bengalensis provoke pancreatic beta pancreatic cell activity. The biomarkers for liver and kid-
cells for production of insulin [20]. Certain plant extracts ney function are creatinine, SGOT, and SGPT, respectively.
have been shown to reduce insulin resistance or reduce According to the findings, STZ caused induced diabetic
hepatic glucose production [21]. Also, the treatment of control rats to have considerably higher serum SGPT and
Panchavalkalakwatha to rats with induced diabetes caused a SGOT levels. This could be as a result of the liver’s cytosol
considerable increase in body weight, which infers indirectly releasing these marker enzymes into the bloodstream, which
from the herb’s possible health benefits. Depletion of body would indicate the liver toxicity of STZ. Diabetes is linked to
proteins is a key indicator of severe body weight loss in dia- liver dysfunction since the liver is the body’s most important
betes mellitus patients. Changes in glucose metabolism have organ and is in charge of maintaining the body’s lipid and
been ascribed to the loss of protein-energy that comes along glucose balance. Necrosis of liver cells, lipid peroxidation,
with high glucose levels [22]. With type 2 diabetes mellitus, and an increase in serum biochemical markers like SGOT
the surrounding cells lose their ability to use glucose as an and SGPT are all signs of hepatic injury [15]. When given
energy source, which causes increased protein intake and to diabetic rats for 28 days at the same dose (10.33 ml/kg
decreased protein storage, both of which result in weight body weight mg/kg), Panchavalkala kwatha kept SGOT
loss in diabetic people. The kidney takes extra sugar from and SGPT levels within normal limits. When compared
the blood and excretes it in the urine when the blood glucose to diabetic control rats, animals with diabetes treated with

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 R. Tripathi et al.

Panchavalkalakwatha and glibenclamide showed a substan- kwatha and glibenclamide treatment of diabetic rats causes
tial (P0.001, P0.01, and P0.05) decrease in SGPT, SGOT, the regeneration of pancreatic cells, possibly via preventing
and creatinine. This shows that the purified fraction has the the formation of free radicals. In Diabetes mellitus, tissue
capacity to normalise liver cells, resulting in the regeneration damage brought on by the production of free radicals is fre-
of parenchymal cells, strengthening the membrane against quently followed by the peroxidation of unsaturated fatty
fragility, and avoiding the leaking of these marker enzymes. acids, which causes membrane damage and dysfunction. Via
Our research strongly revealed that Panchavalkala kwatha anti-oxidant activity, flavanoids found in the aqueous extract
has hepato-protective properties. Due to under consumption of F. religiosa and F. bengalensis raised the level of lipid
of glucose in diabetic patients, excessive fat is mobilised peroxidation. In this manner, it prevents many important
from adipose tissue, which causes hyper-lipidemia. With an organs’ tissue damage [20].
increase in blood sugar, TC, TGs, and VLDL levels rise
while HDL levels fall. These potential outcomes may result
from insulin’s influence on the regulation of lipolysis or from
changes in the activity of the enzymes involved in choles- Conclusion
terol production. Similarly, Panchavalkalakwatha has been
demonstrated to have anti hyper-lipidemic effects while rais- The Panchavalkala kwatha, an Ayurvedic polyherbal for-
ing HDL cholesterol when administered twice day at a dose mulation revealed antihyperglycemic and hypolipidimic
of 10.33 ml/kg body weight. As compared to normal control properties in streptozotocin induced diabetic wister rats. The
rats, diabetic rats generated by STZ had elevated levels of formulation showed cell proliferation in pancreatic β-cells
TG, TC, LDL, and VLDL and dramatically decreased levels with no any sign of toxicity. This study advocate the integra-
of HDL (P 0.001).When compared to diabetic control rats, tion of traditional medicine system with modern medical
diabetic rats treated with both doses of Panchavalkalakwatha sciences as integration of the formulation potentiates the
and glibenclamide demonstrated highly significant increases efficacy of Glibenclamide without inducing any toxicity in
in HDL levels after glibenclamide administration (0.517 mg/ the animal model.
kg b. wt.). Although the percentage of the elevated level is
Acknowledgements The authors are thankful to SAIF, IIT Roorkee,
lower in the group treated with both modern and Ayurveda and Research Facilities of the Department of Dravyaguna, Institute of
formulations than in the group treated with Panchavalkalak- Medical Sciences, Banaras Hindu University, Varanasi.
watha, both groups exhibit substantial raised levels. The rise
in plasma insulin level accounts for the normalised level of Authors’ Contributions RT, RKS, and AKS have designed the study
and completed by RT and RKS, whereas AR and RP helped in writing
lipid cholesterol. The lipoprotein lipases are activated by this
of the manuscript.
increase in plasma insulin, which results in the hydrolysis
of stored TGs into free fatty acids.Reduced TG levels found
in the current study are quite important and demonstrate
how well kwatha works to activate lipase linked to endothe- Data Availability On request, all raw data will be provided.
lium, which hydrolyzes TGs into fatty acids. Moreover, bio-
Declarations
markers for hyperlipidemia and atherosclerosis include TC,
TGs, VLDL, and HDL. As a result, the considerable drop in
Conflict of interest The authors declare that they have no conflict
VLDL and rise in HDL reinforce the active fraction’s ability
of interest.
to lower cholesterol. One of the finest ways to comprehend
the impact of a supplied drug is through histology investi- Consent for Publication We all have confirmed that there are no
gations of key organs. Since the pancreas and liver are the known conflicts of interest associated with this publication.
two main organs that are impacted by diabetes, investiga-
tions on the morphological and physiological changes in the
pancreas have been conducted. According to the data, rats
given diabetes were shown to have a small number of islet References
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