Ethnomedicinal Investigation of Medicinal Plants of Chakrata Region (Uttarakhand) Used in the Traditional Medicine for Diabetes by Jaunsari Tribe

The Himalayan region is the treasure house of natural wealth, particularly of medicinal and aromatic plants. These plants are used by the Indian traditional healers for the past many centuries to treat various ailments such as skin disorders, asthma, diabetes, snake bite, fever, pain, eye diseases, diarrhoea, indigestion, jaundice, burn, wound, liver disorder, CNS disorders and urinary tract infection. The indigenous traditional knowledge of medicinal plants and therapies of various local communities has been lost due to changes in traditional culture and the introduction of modern technologies. Therefore, it is essential to explore the traditional knowledge of the indigenous medicinal plants mainly in such areas where there is a severe threat to natural vegetation owing to human inhabitation. The present study aimed to explore the medicinal plants of Chakrata region (Jaunsar–Bawar Hills), Uttarakhand, India used in the folk medicine for the management of diabetes by Jaunsari Tribe. In a comprehensive field survey, the information about the medicinal plants have been mainly collected from the traditional healers and other elderly people belong to the tribal community. All the information about the medicinal plants of the study area was documented in a field book. Various tools have been used to collect the samples for identification purpose and the authentication of the plants was done with the help of taxonomists. The literature on these plants was also searched from online (PubMed and Scopus) as well as from some textbooks and Ayurvedic classical texts. The present survey-based work described a total of 54 plants belonging to 47 genera and 30 families used in the traditional medicine for the management of diabetes in Chakrata region. The information gathered from the local community revealed that the plants are effective in diabetes and one can use most of them without consulting a practitioner or traditional healer. The literature revealed that most of the surveyed plants are already used in the preparation of various antidiabetic formulations such as Chandraprabha vati, Nishamalaki chunra, Amritamehari churna and Nisakathakadi kashayam along with various patent drugs which are frequently prescribed by the Ayurvedic practitioners in India. The present study explored the traditional as well as scientific knowledge on the antidiabetic plants used by the tribal community. The documented information on these plants can be further used by the scientific community to develop new drugs/formulations with the help of modern techniques.


Introduction
Human has been directly or indirectly depending on the plants since time immemorial to fulfil his daily needs like food, oxygen, medicine and timber. Medicine is one of the essential necessities of a human, and the plants are the primary source of it. The plants have therapeutical importance to treat various kind of human and livestock ailments due to the presence of a variety of bioactive secondary metabolite. In different traditional medicinal systems like Indian and Chinese, these plants are used as a whole or their derived products in the form of different formulations. This ancient system of healthcare is also relevant and effective in the present time when technological progress has been drastically altered the individual's lifestyle.
India is one of the mega-diversity countries recognised by the World Conservation Monitoring Centre in 2000. Its total geographical area is nearly 329 million hectares comprised of forests, grasslands, wetlands, coastal, marine and desert. India has an enormous ecological diversity ranging from sea level to the highest mountains. It represents 2.4% of world's total geographical area with about 47,513 plants species out of 465,668 including microorganisms like virus, bacteria and fungi worldwide. The total numbers of species of angiosperms, bryophytes, pteridophytes and gymnosperms found worldwide are 268600, 16236, 12000 and 1021, respectively [1], whereas, in India, these are 18043, 2523, 1267 and 74, respectively. Among them, 4036, 629, 47 and 8, respectively are reported to endemic to India [2]. In India, about 280 medicinal plants belonging to 79 families are used by pharmaceutical industries to prepare different formulation used in Ayurvedic, Homeopathy, Unani, Siddha and even in Allopathic medicine, of which about 175 medicinal plants are found in the Himalayan region of India [3].
Diabetes mellitus is a metabolic disorder characterised by a high blood glucose level mainly due to the problem of inadequate insulin secretion by the pancreatic β-cells. A continuous high level of glucose in the blood can lead to serious complications such as neuropathy, retinopathy and nephropathy. In addition, one can become prone to heart attack and stroke if the glucose level is not maintained regularly. The patients of this deadly disease have been increased sharply in the past few years and still, the graph is rising rapidly. In 2013, the number of diabetic patients was about 422 million whereas this number was about 108 million in 1980. The cases of diabetes are recorded more in middle and low-income countries than in high-income countries.
In 2015, about 1.6 million deaths were recorded due to diabetes mellitus. The majority of cases belong to type 2 diabetes mellitus, and these are about 80-90% of total diabetic cases. According to the World Health Organization [4], diabetes imputes the largest burden on the global economy and healthcare system. South-East Asia region which includes India, Bangladesh, Maldives, Nepal, Mauritius and Sri Lanka has one fifth (~ 84 million) of the total number of people with diabetes in the world, and hence called as the home of diabetes.

Geography and Culture of the Study Area
Uttarakhand Himalaya is characterised as one of the microdiversity centres of the Indian subcontinent. Chakrata, also known as Jaunsar-Bawar, is a hilly region in district Dehradun situated at a height ranging from 1500 to 2150 m. The Jaunsar region is the lower half while the snow-clad upper region is called Bawar. A tribal community, known as Jaunsari is inhabited in this region, and the native language spoken by them is Jaunsari, although Hindi is also spoken frequently. The culture of Jaunsari people is distinct from other natives of Uttarakhand and Himachal Pradesh, a neighbour hill state. The main occupation of this community is agriculture and animal husbandry. Geographically, it has rich vegetation and mostly covered by forest areas. The region is well-known for the medicinal plant diversity which includes many rare plants [5].
The villages of Chakrata come under remote areas, therefore, less developed community centres and hospitals situated in the region. The insufficient medical facilities make compulsion to the people to use home remedies for all kind of health problems. Local traditional healers or Vaidyas works as the medical practitioner using native herbs based on traditional knowledge. Majority of the population believe in traditional medicine rather than modern treatment until unless a major problem occurs. The geographical location of the study area is shown in Fig. 1.

Data Collection
The literature based on the area, vegetation, population, social customs and culture of the study area has been studied before starting the field visits to the Chakrata region. A comprehensive field survey was conducted in the entire Chakrata region, including Tiuni, Deovan, Sahiya and Lokbar sites during February to March 2018. A total of four visits have been organised to get the maximum advantage of the tribal population to collect information about their plant resources for various purposes, mainly for the treatment of diabetes. The information was collected from the local tribal community, elder people, local traditional healers (Vaidyas) and some employees from the State Forest Department posted in this area. In addition to the general conversations with the informants, the questionnaires were also used to obtain information on the medicinal plants with their local names, parts used, mode of preparation and administration. The plants/parts used for the purpose of diabetes were also collected during the field visits for their taxonomical authentication. The collected plants/parts were kept in the deep fridge (− 20 °C) until their further use. After the identification, the plants/parts were pressed, dried and mounted on the herbarium sheets and preserved in the phytochemistry laboratory for future records.

Data Analysis
During the survey, a total of 54 antidiabetic plants belonging to 47 genera and 30 families have been collected. The identification of the plants was done by Dr. M.R. Uniyal (Materia Medica Expert), Dr. A.K. Agrawal (Taxonomist, Govt PG College, Uttarkashi), Dr. Suresh Chaubey and Dr. R.C. Tiwari (Uttarakhand Ayurved University, Rishikul Campus, Haridwar). The identification also confirmed on basis of the local names of plants mentioned in the local Floras of Uttarakhand and the Himalayas. An in-depth literature search was conducted to confirm the scientific names of the antidiabetic plants. The updated botanical names of all plants were cross-checked from the Plant List, a working list of all plant species [6]. After identification, the scientific literature based on the antidiabetic potential of the medicinal plants was reviewed from the online database (PubMed and Scopus) and textbooks as well as from ancient Ayurvedic texts (Samhitas and Nighantus). Based on the literature survey, selected antidiabetic activity-based information has been included in the text for the scientific validation of traditional knowledge.

Results and Discussion
Based on the field survey, about 120 species of medicinal plants were initially identified in the Chakrata region, of which, 54 plants (belonging to 47 genera and 30 families) were recorded as antidiabetic plants used in the traditional medicine. The highest number of anti-diabetic plants were documented in the family Lamiaceae  Table 1, which contains information about the methods of administration and the selected pharmacological evidence of the antidiabetic plants with well-supported by relevant references. The results showed that most of the plants/parts are used in the form of decoction to treat diabetes.
During the field survey, it has been noted that Artemisia sp., C. pareira, J. adhatoda, S. virginianum and V. negundo are commonly found in roadside areas from Sahiya to Chakrata. On the other hand, B. aristata, M. philippensis and S. glabra are the common plants in light forest areas. T. chebula, T. bellirica and P. emblica are found in edges of fields whereas B. ciliata is available at the rocky areas.
Various preclinical and clinical studies, as shown in Table 1, confirmed the role of surveyed plants in the diabetes mellitus. In addition to the crude extracts, various pure molecules have also been investigated as antidiabetic agents such as alliin, betulin, berberine, gymnemic acid and vasicine. Some mechanism-based studies also unravelled the involvement of different pathways in the antidiabetic activity of crude extracts or isolated molecules (Fig. 2).  (2) Hydro-alcoholic leaf extract (5% of total diet) reduced glucose levels, LDL-C, TC, AST, ALT, and fibrinogen in hyper-cholesterolemic rabbits in a 3 days oral treatment [9] (3) Hydro-alcoholic leaf extract (300 mg/ kg) showed hypoglycemic effect similar to glibenclamide in ALX-induced type 1 diabetic rat [10] (4) Scientifically, it has antidiabetic effect in both humans and animals, and can be suggested for the diabetic patients [11] Carum carvi L.

Kashmirajiraka (S), Krishna
Jeera (A), Kala Jeera (H), Black cumin/Caraway (E) Herb (C)/seeds Essential oil (mainly contains carvone, limonene, anethole and carveol) [12] An infusion of seeds in water is taken orally Aqueous seeds extract (1 g/kg/day) decreased blood glucose level and alleviated the body weight loss of STZ-induced diabetic rat in a 21 days treatment [12] Acanthaceae  (14), vasicinone [15] (1) Juice of leaves is used with an empty stomach (2) A decoction of roots (~ 50 g) with cow milk (125 mL) is taken daily in the morning Ethanol extract of leaves (100 mg/kg/ oral) reduced in blood glucose level in ALX-induced diabetic rats in a 6 days treatment. The results were compared with glibenclamide (5 mg/kg). In addition, it showed a positive effect on the glucose tolerance, glycosylated haemoglobin, serum lipid profiles and body weight of diabetic rats [15]  taken orally with water after breakfast (2) The extract/infusion of Triphala powder is taken with almonds in the morning (1) The extract of dried fruits stimulated basal insulin output and potentiated glucose-stimulated insulin secretion in the clonal pancreatic beta-cell line (BRIN-BD11). It also displayed insulin-mimetic activity and enhanced insulin-stimulated glucose uptake in 3T3-L1 adipocytes. The extract also produced a decrease in starch digestion (in vitro) and inhibited protein glycation at a concentration of 50 mg/ mL [32] (2) The oral administration of methanol extract of dried fruits (100 mg/kg) reduced blood glucose level in ALX induced diabetic rats in an 11 days study [33]  The decoction of aerial parts is used either alone or in combination with the dried fruits of Zizyphus jujube (1) The methanol extract of aerial parts (100 µg/mL), betulinic acid (IC 50 3.49 µM) and betulin (IC 50 4.17 µM) showed protein tyrosine phosphatase 1B (PTP1B) enzyme inhibitory activity in vitro [37] (2) The ethanol leaves extract (1 μg/mL) showed insulin secretagogue activity at a concentration of when tested for insulin release from insulinoma cell line (INS-1 cells) [38] (3) Artemisinin showed trypsin inhibitory activity [36] Artemisia vulgaris Herb (W)/whole plant Essential oil (mainly contains α-pinen, menthol, β-eudesmol and spathulenol) [39] A decoction of leaves with water is used in the morning Oral administration of ethanol leaves extract (250 and 500 mg/kg) for 16 weeks reduced the renal impairment in diabetic nephropathy rats through anti-diabetic, anti-oxidant and protective morphological changes in STZ induced diabetes rats [40] Euphorbiaceae  (2) A herbal tea prepared with leaves is also used (1) Ethanol leaf extract (200 and 400 mg/ kg) showed a blood sugar lowering effect on STZ-induced diabetic rats [24] (2) Hydro-alcoholic leaf extract (250 and 500 mg/kg) showed the antidiabetic effect on STZ and nicotinamide induced diabetic rats [48] Ajuga parviflora The decoction of the whole plant is taken twice a day after meal (1) A crude polysaccharide fraction (50 mg/kg) isolated from the leaves reduced food intake, body weight and fasting glucose levels in db/db mice in a 7 days study [52] (2) An iridoid glucoside (50 mg/kg) isolated from V. negundo leaves reduced the levels of plasma glucose, glycosylated haemoglobin and increased in the levels of insulin and haemoglobin in STZ induced diabetic rats in a 30 days study when compared with the glibenclamide (5 mg/kg) which was used as a positive control. Besides, it increased glycolytic enzymes level and glycogen content and decreased the levels of gluconeogenic enzymes in the liver of diabetic rats [53] (4)(5) or leaves paste (one teaspoon) are useful in diabetes (1) Acetone, ethanol and water extract of leaves showed in vitro α-amylase inhibition at different concentrations range from 1.25 to 10 mg/mL, aqueous extract was found least active with an IC 50 value of 9.15 mg/mL. However, these extracts were found poorly active against α-glucosidase inhibition [59] (2) Oral administration of ethanol leaves extract (200 and 400 mg/kg) showed blood sugar lowering activity in diabetic rats [24] (3) Chloroform leaves extract showed oral glucose tolerance activity and reduced the intestinal glucosidase activity in STZ-induced diabetic mice. It increased glucose-6-phosphate dehydrogenase activity and hepatic, skeletal muscle glycogen content, after 21 days of treatment. The study revealed the regeneration of insulin-producing cells and a corresponding increase in the plasma insulin and c-peptide levels with the treatment [60]  (3) 25 mL of stem juice in an equal amount of water is taken twice a day before the meal The oral administration of aqueous root extract (5 and 7.5 g/kg) caused a reduction in the glucose level of blood and urine of ALX induced diabetic rats. It also decreased hepatic glucose-6-phosphatase and serum acid phosphatase, alkaline phosphatase, and lactate dehydrogenase in diabetic rats [69] Moraceae One cup of tea prepared from leaves (or root/ bark) is used with an empty stomach (1) Ethanol leaves extract (600 mg/kg) has therapeutic effects in STZ induced diabetic rats and can restore the diminished β cell numbers in a 35 days study [74] (2) Fruit polysaccharides fractions showed marked antihyperglycemic and antihyperlipidemic activities and repaired the damaged pancreatic tissues of the diabetic rats [75]  The aqueous decoction (25 mL) of aerial parts is taken in the morning after meal (1) The methanol leaves extract (200 mg/ kg) showed antidiabetic activity against ALX induced diabetic rats [97] (2) Lupeol, isolated from stem bark suppressed the progression of diabetes in rats. Its treatment caused decreasing glycated haemoglobin, serum glucose and nitric oxide in a 21 days study [98] (3) The aqueous fruits extract showed hypoglycemic activity in diabetic rats [99] Taxaceae Taxus baccata The juice or tea prepared from leaves is taken once a day after meal Aqueous leaves extract (300 mg/kg) showed a reduction in the blood glucose level in STZ-induced diabetic rats using a GTT assay [103]  to STZ-induced diabetic rats showed restoration of blood glucose levels with a concomitant increase in insulin levels. The treatment also increased the number, diameter, volume and area of the pancreatic islets of diabetic rats [105] (2) The leaf gel (20, 30 and 50 mg/ mL) and its phytosterols (lophenol, 24-methyl-lophenol, 24-ethyl-lophenol, cycloartanol, and 24-methylenecycloartanol) showed anti-diabetic activity in type 2 diabetic mice. The phytosterols also reduced the HbA1c levels in mice in a 28 days study [104] Zingiberaceae Curcuma longa L. Haridra (S), Haldi (H), Turmeric (E)

Herb (C)/rhizome
Curcumin (22), demethoxycurcumin, bis-demethoxycurcumin, ar-turmerone [106] (1) A small bit of raw rhizomes is chewed empty stomach (2) The powder of dried rhizomes (1 teaspoon) or paste of fresh rhizomes (1 teaspoon) is used in food preparations (1) The volatile oil obtained from the rhizome inhibited α-glucosidase enzymes more effectively than the acarbose (a standard drug) in vitro. A major volatile constituent (ar-turmerone) also had potent α-glucosidase (IC 50 = 0.28 μg) and α-amylase (IC 50 = 24.5 μg) inhibition [107] (2) Curcumin, a phenolic compound isolated from the rhizome reduced blood glucose and the levels of glycosylated haemoglobin in diabetic rats through the regulation of the polyol pathway. It suppressed oxidative stress and inflammation, and also suppressed increased bone resorption through the inhibition of osteoclastogenesis and expression of the AP-1 transcription factors, c-fos and c-jun, in diabetic animals. It showed a beneficial role in the diabetes-induced endothelial dysfunction and induced a down-regulation of nuclear factor-kappa B. It has a protective role against advanced glycation as well as collagen cross-linking [108] [112] (2) The oral dose of essential oil obtained from rhizomes (0.3 mL for 14 days) reduced blood glucose and urea level in ALX induced diabetic rats. It was also observed that the Islets of Langerhans regained their normal shape after 14 days of treatment [113] Zingiber officinale  [116]. Kachnar (B. variegata), which is used in wounds, ulcer, thyroid problems, cervical lymphadenitis and rectal prolapse, is an important ingredient of Kachnar guggulu, Chitrakadi taila, Chandanasava, Ushirasava, Gandamala kandana rasa and Mutra sangrahaniya kwata. According to the National Medicinal Plants Board, Ministry of AYUSH, Government of India, its current market demand is also about 100-200 metric tons annually. Talispatra (T. baccata) and Devadaru (C. deodara) are also important Ayurvedic medicinal plants and their annual market demand is 100-200 and 1000-2000 metric tons, respectively.

Mechanism of Actions of Surveyed Plants and Their Bioactives
Various earlier research studies reported the possible mechanisms of action shown by the antidiabetic plants as well as their bioactive constituents described in the present paper. A. vera and its phytosterols, i.e. lophenol, 24-methyl-lophenol, 24-ethyl-lophenol, cycloartanol and 24-methylene-cycloartanol shows glucose lowering effect through effectively decrease gluconeogenesis and lipogenesis processes. This activity predominantly appears to be mediated by AMPK and PPAR receptors and is accompanied mainly by inhibition of PEPCK and G6P genes expression or inhibition of ACC and FAS enzymatic activities hence reduce hepatic glucose output [104]. Moreover, the hypoglycemic effect of aloes and other bitter principles may be mediated through stimulating synthesis and/or release of insulin from the β-cells of Langerhans [117]. Berberine, an active principle of B. aristata was found to alter glucose metabolism through the stimulation of glycolysis via increasing glucokinase activity, increasing insulin secretion, and suppressing hepatic gluconeogenesis and adipogenesis [118][119][120]. Similarly, a bioactive molecule S-methycystein sulphoxide found in A. cepa regulates the enzyme Glucokinase/Hexokinase and stimulates glucose utilization and insulin secretions [121]. On the other hand, quercetin, another bioactive of A. cepa increases glucosestimulated insulin secretion through an ERK1/2 pathway which improves liver and pancreas functions by enabling the recovery of cell proliferation through the inhibition of Cdkn1a expression [122].
Curcuma longa and its active molecule curcumin increase the islet viability and delay islet ROS production in animals [123]. Curcumin treatment also increases the number of small pancreatic islets and decreases lymphocyte infiltration in pancreatic islets [124]. The intervention of a curcumin-rich extract improves β-cell functions, indicated by an increased HOMA-β and reduced C-peptide in a study conducted by Chuengsamarn et al. [125]. The molecule itself plays antioxidant defence by induction of the expression of HO-1, glutathione subunit, and NAD(P)H:quinone oxidoreductase 1 and increased basal insulin secretion in human islet [126].
Artemisia roxburghiana as well as its bioactives, such as artemisinin, betulinic acid and betulin, act as protein tyrosine phosphatases inhibitor. Through this mechanism, they are known to enhance insulin receptor phosphorylation and stimulate glucose uptake into cells. On the other hand, its flavonoids inhibit cAMP phosphodiesterase which is a modulator of insulin secretion [37]. G. sylvestre has been reported to interact with glyceraldehyde-3-phosphate dehydrogenase, a key enzyme in the glycolysis pathway. In addition, its extract stimulates insulin secretion from human islets [127] and regenerates pancreatic β-cell in diabetic animals [128]. A leaves extract containing gymnemic acids as a bioactive suppresses the elevation of blood glucose level by inhibiting glucose uptake in the intestine [129]. Gulfraz et al. [15] reported that J. adhatoda extract increases the level of insulin in diabetic rats. It brings antihyperglycaemic action perhaps by potentiation of pancreatic secretion of insulin from β-cell of islets or due to enhanced transport of blood glucose to peripheral tissue.
Morus alba has found to possess antihyperglycemic and antihyperlipidemic activities by activating the IRS-1/PI3K/ Glut-4 signalling pathway in skeletal muscles and hence increases peripheral glucose uptake [130]. It interferes with the activity of α-amylase that exhibited an effective strategy to lower the levels of hyperglycemia through the control of starch breakdown [131]. In addition to inhibiting α-amylase, it reduces glucose level via scavenging of ROS and regenerating β-cell [132]. M. azedarach decreases blood glucose via inhibition of protein tyrosine phosphatase 1B (PTP1B) enzyme activity and stimulation of glucose uptake rats [133]. T. sinensis was also found to inhibit PTP1B activity and stimulate the glucose uptake [134]. R. australe and its active molecules rhapontigenin, desoxyrhaponticin and desoxyrhapontigenin inhibit the mammalian α-glucosidase [135], whereas V. negundo inhibits α-amylase to decrease blood glucose level [136]. T. foenum-graecum stimulates insulin secretion at all levels of the cellular organization since the cells are more sensitive to insulin and increase in the number of insulin receptor sites to burn cellular glucose at high fibre diet in rats [137]. A schematic diagram of the antidiabetic mechanisms of selected plants/bioactives is shown in Fig. 3.

Status of the Medicinal Plants of Chakrata Region
Various plants in the Chakrata region are rare and only grown as wild, their cultivation is still a big challenge. The surveyed plants were checked in the IUCN Red List [138] for their current status and found that many of them are already included in the list of threatened species. According to the IUCN List, B. variegata, C. deodara, T. baccata and U. dioica are identified as the plants of least concern whereas A. heterophyllum and S. hexandrum are reported as an endangered plant. Although many rare plants, including A. heterophyllum, are cultivated in a big scale in India and exported to other countries [139], many other plants of this particular region are also in the stage of vulnerable and their protection is highly needed. Various reports published in past years also highlighted the importance of these plants including their medicinal uses, market demand and current status [140][141][142][143].

Conclusion
The Chakrata region (Jaunsar-Bawar Hills) is one of the sites of the rich diversity of medicinal plants in Uttarakhand, India. Most of the areas of this region are covered by the forests with a very scattered population mainly the tribal community. The population is mainly depends on folk medicine due to limited hospitals in the region. These people prefer to consult the local traditional healers or elderly person in the family for the treatment, and only moved to the distantly located hospitals in case of any serious health trouble. There are various nurseries, such as Deovan and Khoonigarh, in the region established by the Forest Department (Govt. of Uttarakhand) to cultivate valued medicinal and other economic plants including some fruits and timber yielding plants. In addition, the natives also cultivate several There is good support of the local government to encourage them by providing quality seeds, fertilisers and tools in subsidised rates.
The present study site has numerous antidiabetic plants found as wild such as B. aristata, J. adhatoda, C. pareira, S. glabra, A. roxburghiana, T. baccata, etc. This region is also popular for cultivating many rare medicinal plants such as A. heterophyllum, S. hexandrum and B. variegata. Thus, with the help of cultivation in a big scale, the threatened plants in this region can be conserved for the future. The overharvesting of the medicinal plants can also be minimised by investigating their bioactive compounds and synthesising them in the laboratories to fulfil the market demand of such plants. This approach may also be worthy in case a particular medicinal plant becomes extinct in the future, its drug/ formulation will be always available for the treatment of a particular disease.