ETHNOMEDICINAL VALUES AND ANTIDIABETIC POTENTIAL OF CLERODENDRUM SPP. OCCURRING IN NORTHEASTERN REGION

ETHNOMEDICINAL VALUES AND ANTIDIABETIC POTENTIAL OF CLERODENDRUM SPP. OCCURRING IN NORTHEASTERN REGION

Janmoni Kalita ^{* 1}, S. Sureshkumar Singh ² and Mohamed Latif Khan ³

Department of Biotechnology ¹, Gauhati University, Guwahati - 781014, Assam, India.

Department of Forestry ², North Eastern Regional Institute of Science and Technology (Deemed University), Nirjuli - 791109, Arunachal Pradesh, India.

Department of Botany ³, Dr. Harisingh Gour Central University, Sagar - 470003, Madhya Pradesh, India.

ABSTRACT: The present study was undertaken to systematically analyze, document the traditional knowledge of Clerodendrum species use for the treatment of various human ailments from NER. The information was collected by literature survey as well as by consulting questionnaire with the villagers and local communities of NER. A total of twelve Clerodendrum species were collected, and among them, C. colebrookianum, C. indicum, C. viscosum were found to have multi-medicinal values that were widely used for the treatment of various diseases. Further antidiabetic properties of Clerodendrum species were evaluated by α-amylase and α-glucosidase assay. The result showed 4 species, C. indicum, C. japonicum, C. serratum, C. viscosum exhibited significant (>50%) α-amylase inhibition properties while 3 other species, C. colebrookianum, C. inerme, C. viscosum displayed significant α-glucosidase inhibition properties. Lowest IC₅₀ values were observed in C. serratum of α-amylase (45µg/ml) and C. viscosum of α-glucosidase (47 µg/ml). A detailed scientific investigation of the biochemical compounds and metabolites with potential biological activities in Clerodendrum species may lead to the discovery of potential drug candidates against life-threatening diseases and will help in scientific understanding and proper utilization in traditional and modern health care systems of the country in particular and the world in general.

Clerodendrum, Diversity, Ethnobotany, Tribal communities, Northeastern region

INTRODUCTION: Clerodendrum is a very large and medicinally important genus reported to have more than 400 species distributed in tropical and subtropical regions of the world ¹. There are about 23 species being reported in India where 16 species and 1 variety are reported from the state of Arunachal Pradesh alone ².

These plants are found abundantly growing in northeastern region (NER) and widely used continuously by the local peoples for treatment of many diseases such as anti-microbial, anti-helmintic, anti-inflammatory, anti-malarial, anti-diabetic, hepatoprotective, indigestion, high blood pressure, high fever, asthma, etc. Phytochemical study of Clerodendrum species reported to have major compounds of alkaloids, phenolics, flavonoids, steroids etc. and various biological activities like antibacterial, antimicrobial, insecti-cidal, antihypertensive, antioxidant etc. have been reported ¹.

Type II diabetes (non-insulin-dependent diabetes mellitus) is a chronic endocrine disorder characterized by hyperglycemia in which blood sugar levels are elevated either because the pancreas do not produce enough insulin or cells do not respond to the produced insulin ³. In the recent past, in-vitro screening of herbal-based inhibitors for alpha (α-) amylase and α-glucosidase enzymes have been important approaches to researchers for the discovery of antidiabetic drugs. α-amylase is the enzyme that hydrolyses the polysaccharide (starch) to oligosaccharides (maltose), and α-glucosidase catalyzes the final step to release the absorbable glucose. Hence, the inhibition of these enzymes leads to a decrease in blood glucose level, which plays an important role in the management of diabetic complications⁴. Since these enzymes play key roles in digestion and intestinal absorption of sugar in the diet, their inhibitors are the potential targets in the development of lead compounds for the treatment of diabetes ⁵. In-vitro screening of α-amylase and α-glucosidase enzyme inhibitors have been reported from many plants including some species of Clerodendrum such as C. bungei, C. multiflorum and C. volubile ^{6, 7, 8} and other plants namely, Artocarpus altilis, Artocarpus heterophyllus, Berberis aristata, Cassia auriculata, Cinnamomum zeylanicum, Piper betel, Terminalia arjuna etc.^{9, 10}

Therefore, the present investigation was carried out to document on the ethnobotany of North East Clerodendrum species and evaluation of an antidiabetic property by in-vitro α-amylase and α -glucosidase assay.

MATERIALS AND METHODS:

Study Area, Species Identification and Herbarium Preparation: Field surveys (2011-2015) were conducted in six states of NER (Arunachal Pradesh, Assam, Manipur, Meghalaya, Nagaland, Mizoram) and Clerodendrum species were collected, identified by examining the morphological and reproductive features with consulting various available floras ^{11, 12}, Botanical Survey of India, Itanagar and available identifying keys ^{13, 2}. The plant list (http://theplantlist.org) was used for the valid names. Each of the herbarium was given a specific voucher number and was deposited in the Herbarium of Department of Forestry, NERIST, Nirjuli, Arunachal Pradesh.

Field Survey and Ethnobotany of Clerodendrum Species from NER: All peer-reviewed journals, book chapters, particularly on ethno-medicinal uses of Clerodendrum species from NER,, were selected for this review and collected by searching major scientific electronic databases including Google Scholar, PubMed, Science Direct, etc.

In terms of journal contribution to this review, the Journal of Traditional Knowledge (13nos.) and Journal of Economic and Taxonomic Botany (11nos.) provided the majority of articles carrying information on the ethnobotany of Clerodendrum species from NER. Further validation was done by making field trips to village areas of tribal communities, and information on medicinal uses of Clerodendrum species was collected through modified semi-structured questionnaire ¹⁴ (Appendix 1). A total of 100 informants were selected at random during house-to-house surveys. The knowledge of medicinal plants >30 years of age was taken into consideration.

Preparation of Plant Material for Bioassay: A total of the twelve Clerodendrum species were collected during the field survey. However, only seven (7) species viz. C. colebrookianum (CC), C. inerme (CIN), C. indicum (CI), C.  japonicum (CJ), C. philippinum (CP), C. serratum (CS) and C. viscosum (CV) were selected for antidiabetic assay based on their medicinal values and availability of plant samples in wild habitats. Young, tender, and disease-free leaves of selected seven Clerodendrum species were rinsed with tap water followed by drying in a hot-air oven (60 °C) for 3-12 h, grounded into powder by using a kitchen grinder.

Preparation of Crude Methanol Leaf Extract: Dry powdered leaves (1kg) of seven Clerodendrum species were suspended in methanol (ME) and kept overnight in a rotary shaker (Scigenics Biotech, Chennai) at 100 rpm. The slurry was filtered on sterile Whatman filter paper (110mm), and the green-colored filtrate was vacuum dried (below 25 °C) in a rotary evaporator (RV-10, IKA, Germany). The yield of the extracts was recorded, and the samples were finally stored at -20º separately. The crude methanol extracts were screened for their inhibitory activities against α-amylase and α-glucosidase.

Chemicals and Reagents: α-amylase ex-porcine (SRL148188), α-glucosidase (Maltase) (SRL74854), p-nitrophenyl α-D-glucopyranoside (SRL144969) were purchased from SRL India. Acarbose (A8980) was purchased from Sigma. Starch, NaOH pellets, Na₂HPO₄, NaH₂PO₄, NaCl, calcium chloride, dimethyl sulphoxide, Tris base, and other chemicals and solvents were of highest purity grade and purchased from Merck, Mumbai, India. Milli-Q water was used for all the enzymatic assays.

Preparation of Enzyme, Substrate, and Buffer Solutions for α-amylase Assay:

Incubation Buffer: 0.05M Tris Hcl buffer (pH 6.9) containing 0.01M CaCl_2.

Enzyme Preparation (20U/ml): A stock solution of 1mg/ml (1mg=20U) α-amylase prepared in Tris-HCl buffer.

Substrate Starch Solution: Starch azure 0.1% in 0.05M Tris-Hcl buffer (pH 6.9) containing 0.01M calcium chloride and boiled for 5-10 min to properly dissolve starch in solution.

Positive Control Acarbose: A stock solution (1mg/ml) prepared in distilled water (Millipore Type I).

Test Sample: A stock solution (1mg/ml) prepared in DMSO and further diluted with water upto 1ml. DMSO concentration was below 2%.

Terminating Solution: 50% acetic acid.

Color Reagent: Iodine solution was prepared by dissolving 0.254g I₂ and 4g KI in 1L of distilled water.

Preparation of Enzyme, Substrate, and Buffer Solutions for α-glucosidase Assay:

Incubation Buffer: 0.1M phosphate buffer, pH 6.8

Enzyme Preparation (64U/ml): A working stock of 1mg/ml (1mg=64U) α-glucosidase prepared in phosphate buffer.

Substrate Preparation: 0.5mM of p-nitrophenyl α-D glucopyranoside (PNPG) prepared in phosphate buffer.

Positive Control Acarbose: 1mg/ml dissolved in distilled water (Millipore Type 1).

Test Sample: A stock solution (1mg/ml) prepared in DMSO and further diluted with water upto 1ml. DMSO concentration was below 2%.

Terminating Solution: 0.2M sodium carbonate solution.

α-amylase Inhibition Assay of Crude Leaf Extract: In-vitro α-amylase enzyme inhibition assay was done by following partial modification of the starch-iodine method ^{15, 16}.

A volume of 200µl starch solution (0.1% starch in 0.05M Tris HCl buffer containing 0.01M CaCl₂) was taken in a test tube (10 × 100mm) and pre-incubation at 37 °C for 5 min. After pre-incubation, a sample extract solution of 25µl (1mg/ml), 5µl of the α-amylase enzyme (1U) and 270µl of buffer were added to the test tube to make the final reaction mixture of 500µl. The reaction tube was further incubated at 37 °C for 10minutes and stopped by adding 500µl of 50% acetic acid. The reaction mixture was then centrifuged at 3000 rpm for 5 min at 4°C. The upper centrifuged part of the reaction mixture was transferred into a clean and dry test tube. The reaction mixture was allowed to develop color by adding 1ml of iodine solution followed by vortexing for 30 sec. The absorbance was measured at 565nm in a spectrophotometer (Multiskan GO, Thermo-Scientific, Finland). Acarbose was used as standard inhibitor drug for α-amylase. Appropriate sample blanks and controls were included for each sample treatment. All reactions were performed in triplicate. The inhibition (%) of α-amylase activity was calculated by using the formula.

α -amylase inhibition (%) = Sample OD × 100/ Control OD

[Sample OD= Sample OD- Sample blank OD; Control OD = OD of the control reaction without inhibitor-blank OD].

Dose-Response Analysis of α-amylase Inhibition Assay: The significant Clerodendrum species (showed >50% inhibition) were further selected for dose-dependent analysis by taking different con-centrations i.e., 10-100µl≈20-200µg/ml, respectively.

α-glucosidase Inhibitory Assay of Crude Leaf Extract: The α-glucosidase enzyme inhibitory assay was performed in a chromogenic method in which enzyme activity was estimated by measuring yellow color developed due to release of p-nitrophenyl after cleavage of glycosidic linkage of substrate p-nitrophenyl α-D glucopyranoside (PNPG)⁴. 25μl of sample solution was premixed with 25μl of the enzyme (0.5U) and incubated at 37 °C ± 1 °C for 10 min. After incubation, 25μl of the substrate (0.5mM, p-nitrophenyl α-D glucopyranoside) was added to the reaction mixture and incubated at 37 °C ± 1 °C for 30 min. The reaction was terminated by adding 100μl of 0.2M sodium carbonate solution. The amount of p-nitrophenol released from PNPG was quantified on a 96 well microplate at 405nm in a microplate reader (Multiskan GO, Thermo-Scientific, Finland). Appropriate sample blanks and controls were included for each sample treatment. Acarbose was used as a standard inhibitor drug for α-glucosidase. All reactions were performed in triplicate. The inhibition (%) of α-glucosidase activity was calculated by using the formula.

α-glucosidase inhibition (%) = Control OD-Sample OD × 100/ Control OD

[Control OD = OD of the control reaction without inhibitor-blank OD; Sample OD = Sample OD- Sample blank OD].

Dose-Response Analysis of α-glucosidase Inhibition Assay: The significant Clerodendrum species (showed >50% inhibition) were further selected for dose-dependent analysis by taking different concentrations i.e. 5µl to 30µl≈67µg/ml to 400µg/ml respectively.

IC₅₀ Calculation: The half-maximal inhibitory concentration (IC₅₀) is a measure of the effectiveness of a substance in inhibiting a specific biological or biochemical function. IC₅₀ values were calculated by using an online tool (www.ic50.tk) from the dose-response curve for both α-amylase and α-glucosidase assay.

RESULTS:

Diversity and Ethnobotany of Clerodendrum Species in NER: Present investigation on the taxonomic diversity of Clerodendrum species revealed the rich diversity with the occurrence of 12 numbers of species (C. bracteatum, C. colebrookianum, C. inerme, C. indicum, C. japonicum, C. philippinum, C. serratum, C. speciosum, C. splendens, C. thomsonii, C. viscosum and C. wallichii) from NER Table 1, Fig. 1.

Literature survey covered a total of 60 numbers of reports particularly on ethnomedicine of Clerodendrum species resulted in ten species representing 37 diseases used by 26 tribal communities in entire NER while field survey analysis recorded 9 Clerodendrum species representing 16 diseases used by 13 tribal communities of NER. In contrast, the single species, C.thomsoniae was found to be used as ornamental Table 2.

 TABLE 1: DETAILS OF TWELVE CLERODENDRUM SPECIES, THEIR VERNACULAR NAMES, AND PLACE OF COLLECTION FROM NORTHEASTERN REGION

AR-Arunachal Pradesh; AS-Assam; MN-Manipur; MZ-Mizoram; ML-Meghalaya; NL-Nagaland

FIG. 1. CLERODENDRUM SPECIES IN NATURAL HABITAT (1) C. BRACTEATUM (2) C. COLEBROOKIANUM (3) C. INERME (4) C. INDICUM (5) C. JAPONICUM (6) C.  PHILIPPINUM (7) C. SERRATUM (8) C. SPECIOSUM (9) C. SPLENDENS (10) C. THOMSONII (11) C. VISCOSUM (12) C. WALLICHII

TABLE 2: CLERODENDRUM SPECIES USED IN DIFFERENT AILMENTS AND DISEASES BY VARIOUS TRIBES OF NORTHEASTERN REGION (LITERATURE SURVEY REPORT)

B- Bark; F- Flower; L- Leaf; R- Root; S- Seed; S-Stem; T-twig; SH-Shoot; WP- Whole plant; TW-Twig. AS-Assam; AR-Arunachal Pradesh; MN-Manipur; MZ-Mizoram; ML-Meghalaya; NL-Nagaland; TR-Tripura; SK-Sikkim.

Among the species, C. colebrookianum has been found to be used in a total of 17 different diseases where a maximum of 21 northeast tribes used for curing of blood pressure, 7 tribes used for diabetes, 4 tribes used for rheumatism, 3 tribes used for diarrhea and dysentery, fever respectively reported from a literature survey. While in field survey, the species has been used in 13 different diseases where a maximum number of informants (90) belonging to 12 different tribal communities used for controlling of blood pressure, 4 tribes used in gastric trouble, fever, dysentery and abdominal pain, 3 tribes in stomach trouble, diabetes, heart trouble, headache and cough, 2 tribes in malaria, 1 tribe in jaundice and sinusitis respectively Table 3. Young and tender leaves were used in the majority of diseases and ailments in both literature and field survey Fig. 2. Mode of administration was found to be leaf boil in both literature and field survey Fig. 3.

FIG. 2: DIFFERENT PLANT PARTS OF CLERODENDRUM SPECIES USED IN TRADITIONAL MEDICINE (LITERATURE SURVEY AND FIELD SURVEY)

FIG. 3: DIFFERENT WAYS OF HERBAL PREPARATION OF CLERODENDRUM SPECIES USED IN TRADITIONAL MEDICINE (LITERATURE SURVEY AND FIELD SURVEY)

TABLE 3: ETHNO MEDICINAL USES OF CLERODENDRUM SPECIES BY VARIOUS TRIBAL COMMUNITIES OF NORTHEASTERN REGION (FIELD SURVEY REPORT)

F- Flower; FR-Fruit; L- Leaf; R- Root, AS-Assam; AR-Arunachal Pradesh; MN-Manipur; MZ-Mizoram; ML-Meghalaya; NL-Nagaland

Screening of α-amylase Inhibition Properties: The crude ME of four species (C. serratum, C. japonicum, C. viscosum and C. indicum) have shown significant inhibition of enzyme activity (>50%) while the other 3 species (C. philippinum, C. colebrookianum, and C. inerme) have shown insignificant inhibition activity (<50%) as compared to the standard inhibitor of α-amylase, acarbose (ACB) (80%) Fig. 4.

FIG. 4: α-AMYLASE INHIBITION PROPERTIES OF SEVEN CLERODENDRUM SPECIES AND STANDARD DRUG ACARBOSE. T-BARS ON THE HISTOGRAM REPRESENT STANDARD DEVIATION (SD)

Dose-Response Analysis of α-amylase Inhibition Assay: The inhibition was found to be increased with increasing concentrations in all species with the lowest inhibition by C. serratum (28% in 10µl) to highest inhibition by C. indicum and C. serratum (97% in 100µl) respectively. The inhibition activity for standard drug acarbose (ACB) was between 7 to 85% in 10 to 100µl concentrations Fig. 5.

FIG. 5: COMPARISON OF α-AMYLASE INHIBITION PROPERTIES OF FOUR CLERODENDRUM SPECIES AND STANDARD DRUG ACARBOSE (ACB) AT DIFFERENT CONCENTRATIONS (10-100µL≌20-200µG/ML).T-BARS ON THE HISTOGRAM REPRESENT STANDARD DEVIATION (SD)

Screening of α-glucosidase Inhibition Properties: The crude ME of 3 species were found to possess significant inhibition (>50%) of α-glucosidase activity. C. viscosum was found to show the highest level of inhibition (73%) followed by C. inerme (68%) and C. colebrookianum (59%) in comparison with standard drug, acarbose (86%). Four species (C. japonicum, C. serratum, C. philippinum and C. indicum) showed no significant inhibition of the enzyme activity Fig. 6.

FIG. 6: α-GLUCOSIDASE INHIBITION PROPERTIES OF SEVEN CLERODENDRUM SPECIES AND STANDARD DRUG ACARBOSE. T-BARS ON THE HISTOGRAM REPRESENT STANDARD DEVIATION (SD)

Dose-Response Analysis of α-glucosidase Inhibition Assay: The α-glucosidase inhibition percentage was found to be increased with increasing concentrations of the leaf extract. C. viscosum showed the highest inhibition in all concentrations (23% in 5µl to 83% in 30µl). Similarly, C. colebrookianum and C. inerme also displayed enzyme inhibition properties between the lowest of 18% (5µl) to a maximum of 80% (30µl), respectively Fig. 7.

FIG. 7: COMPARISON OF α-GLUCOSIDASE INHIBITION PROPERTIES OF 3 CLERODENDRUM SPECIES AND STANDARD DRUG ACARBOSE AT DIFFERENT CON-CENTRATIONS (5-30µL ≈ 67-400µg/mL). T-BARS ON THE HISTOGRAM REPRESENT STANDARD DEVIATION (SD)

50% Inhibition Concentration (IC₅₀) of Leaf Extracts: The IC₅₀ values showed ranged from a minimum of 45µg/ml of C. serratum to a maximum of 71µg/µl of C. japonicum as compared to positive control acarbose (79µg/ml) for α-amylase. On the other hand, IC₅₀ values for α-glucosidase ranged from 47µg/ml of C. viscosum to a maximum of 184µg/ml of C. inerme as comparison to acarbose (244µg/ml). Among the species, crude ME of C. serratum and C. viscosum showed the lowest IC₅₀ values of α-amylase (45µg/ml) and α-glucosidase (47µg/ml) Table 4.

TABLE 4: IC₅₀ VALUES OF LEAF EXTRACTS WITH POTENTIAL Α-AMYLASE AND Α-GLUCOSIDASE INHIBITION PROPERTIES FROM CLERODENDRUM SPECIES. VALUES WITHIN PARENTHESIS SHOW SD OF MEAN

DISCUSSION: From the present observation, it has been found that many Clerodendrum species growing in NER are continuously used by tribal communities for the treatment of many diseases in the form of special preparations or as vegetables. C. colebrookianum, C. inerme, C. indicum, C. serratum were found to have multi medicinal properties. To validate the traditional claims associated with Clerodendrum species, many scientific investigations were carried out by using in-vitro and in vivo assays. In the present study, 4 species of Clerodendrum (C. serratum, C. japonicum, C. viscosum, C. indicum) exhibited significant α-amylase inhibition properties while 3 other species (C. viscosum, C. colebrookianum, C. inerme) displayed significant α-glucosidase inhibition properties.

Previous studies have reported crude ME of C. viscosum possessed hypoglycemic property against streptozotocin and alloxan-induced diabetes in wister rats ^{17, 18}. C. serratum and C. inerme leaf extract were reported to have significant blood glucose-lowering potential in STZ-induced diabetic rats ^{19, 20, 21}. These findings are in agreement with significant in-vitro α-amylase, and α-glucosidase inhibition properties of the methanol extracts of the three species (C. serratum, C. viscosum and C. inerme) reported in the present study.

The present study of C. colebrookianum methanol leaf extract was found to show moderately high percentage inhibition of α-glucosidase activity. A study on experimentally induced insulin resistance rats also revealed significant ameliorating role of aqueous leaf extract of C. colebrookianum ²². C. colebrookianum, C. indicum, C. inerme, C. japonicum, C. philippinum, C. viscosum and C. serratum are reported to contain various bioactive compounds such as acacetin, acteoside, apigenin-6-C-β-l-fucopyranoside, apigenin-7-0-glucoside, isoacteoside, isoquercetin, hispidulin, hispidulin 7-O-glucuronide, kaempferol, luteolin, oleanic acid, quercetin, rutin, etc. ^{23, 24, 25, 1}.

All these compounds isolated from other medicinal plants reported having antidiabetic properties; however isolation, particularly from Clerodendrum species together with antidiabetic properties, has not been established.

CONCLUSION: Therefore, it is concluded that four Clerodendrum species were found to possess highly significant antidiabetic properties as revealed by in-vitro α-amylase, and three other species have shown significant α-glucosidase inhibition properties. Despite large information available on phytochemical compounds and their correlation with antidiabetic properties from different species of the genus Clerodendrum, there is scarce information on the phytochemical characterization of the above species with potential antidiabetic properties reported in this study. A further investigation into phytochemical profiling and evaluation of their role in hypoglycemic activities of selected species, specially C. viscosum and C. serratum under in-vitro and in-vivo conditions may help in the identification of lead molecule for therapeutic use in treatment and management of diabetes.

The hypoglycemic effect of Clerodendrum species may be due to the presence of more than one antidiabetic bioactive compound or their synergistic properties. Further bioassay-guided fractionation and in-silico screening bioactive compounds from Clerodendrum species may serve as target inhibitors in the treatment and management of diabetes mellitus in the near future.

ACKNOWLEDGEMENT: The authors gratefully acknowledge the financial support and infrastructure facility of DBT, Govt. of India in the form of NER-Twinning project (HC-118, 2011) & Institute level Biotech HUB (IBTHub), Dept. of Forestry, NERIST (Deemed University), Nirjuli, Arunachal Pradesh. Also grateful to Botanical Survey of India, Arunachal Pradesh for identification of Clerodendrum species and thank local guides, villagers of NER for cooperation, and sharing the valuable information on medicinal uses together with support during the fieldwork.

CONFLICTS OF INTEREST: Nil

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    Kalita J, Singh SS and Khan ML: Ethnomedicinal values and antidiabetic potential of Clerodendrum spp. occurring in Northeastern region. Int J Pharm Sci & Res 2020; 11(10): 5112-24. doi: 10.13040/IJPSR.0975-8232.11(10).5112-24.

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