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Year : 2020  |  Volume : 13  |  Issue : 3  |  Page : 258-263  

Anatomical and antihyperglycemic activity of Dichrostachys cinerea roots

1 Department of Pharmacognosy, Central Ayurveda Research Institute for Drug Development, CCRAS, Ministry of AYUSH, Kolkata, West Bengal, India
2 Department of Pharmacognosy, G. Pulla Reddy College of Pharmacy, Mehdipatnam, Telangana, India
3 Department of Technology, Faculty of Pharmacy, University College of Chemical Technology, Osmania University, Hyderabad, Telangana, India

Date of Submission28-Mar-2019
Date of Acceptance22-May-2019
Date of Web Publication3-Jun-2020

Correspondence Address:
Rajesh Bolleddu
Research Officer (Pharmacognosy), Department of Pharmacognosy, Central Ayurveda Research Institute for Drug Development, CCRAS, Ministry of AYUSH, Government of India, Kolkata - 700 091, West Bengal
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/mjdrdypu.mjdrdypu_95_19

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Background: Dichrostachys cinerea Wight. and Arn. of Mimosaceae family commonly called as Veerataru in Ayurveda. Traditionally, it has been used in diabetes, rheumatism, urinary calculi, renal problems, and menstrual disorders. The plant has been reported to possess diuretic, antiprotozoal, antihelmintic, anticancer, antibacterial, antidiarrheal, and hepatoprotective activity, etc., Although it is an important plant, till date, no reported anatomical, chromatographic data are available on roots. Aim: The present study is aimed to establish anatomical characters of fresh roots of D. cinerea followed by antihyperglycemic and chromatographic studies of various fractions of hydroalcoholic extract of roots. Materials and Methods: Morphoanatomical studies were carried out according to the quality control standards of Ayurvedic Pharmacopoeia of India. Hydroalcoholic extract was subjected to fractionation with nonpolar-to-polar solvents. Antihyperglycemic studies were carried out by glucose tolerance test. Results: Transverse section of the fresh root shows the presence of suberized cork, parenchymatous cells containing numerous starch grains. Ethyl acetate fraction has produced significantly (P < 0.01) highest antihyperglycemic activity (52.32%) at a dose of 400 mg/kg among all fractions. High-performance thin-layer chromatography analysis also confirmed the presence of phenols in ethyl acetate and butanol fractions. Conclusion: The current anatomical and chromatographic analysis can be considered as reference standards for future studies on D. cinerea roots. A traditional claim on antidiabetic activity of roots has been scientifically established.

Keywords: Antidiabetic activity, Dichrostachys cinerea, fractionation, high-performance thin-layer chromatography, starch grains

How to cite this article:
Bolleddu R, Venkatesh S, Hazra K, Rao MM, Shyamsunder R. Anatomical and antihyperglycemic activity of Dichrostachys cinerea roots. Med J DY Patil Vidyapeeth 2020;13:258-63

How to cite this URL:
Bolleddu R, Venkatesh S, Hazra K, Rao MM, Shyamsunder R. Anatomical and antihyperglycemic activity of Dichrostachys cinerea roots. Med J DY Patil Vidyapeeth [serial online] 2020 [cited 2021 Aug 4];13:258-63. Available from: https://www.mjdrdypv.org/text.asp?2020/13/3/258/285775

  Introduction Top

Dichrostachys cinerea Wight. and Arn.(Family-Mimosaceae) is a small tree, occurring in the dry scrub forest and arid hills of Northwestern, Central, and Southern India. It grows up to 2 m in height. Its roots are traditionally used in rheumatism, renal troubles, urinary calculi, in vaginal and menstrual disorders.[1] In Ayurveda, D. cinerea plant is used as asmari, mutrakrcchra, mutraghata, trsna, yoniroga, and sandhisula.[2] The methanolic extract of root at 200 mg/kg dose significantly protected mice against the lethal effect (47.5 μg/20 g) of Russell's viper venom when administered 1 min after the venom.[3] Antidiarrheal activity of ethanolic extract is reported at 200 and 400 mg/kg dose.[4] Diuretic property observed when the roots are administered orally in the form of Kwatha at a dose of 5.4 and 10.8 ml/kg.[5] Root bark reported to possess mild anticonvulsant activity.[6] β-sitosterol, β-amyrin, and n-octacosanol were isolated from benzene extracts.[7] Eighteen new monoterpene derivatives (Dichrostachins; A-R) isolated from the root and stem bark.[8]D. cinerea aerial parts also reported to possess epicatechin,[9] mesquitol,[10] anthocyanins, and anthoxanthins.[11] The D. cinerea roots decoction is claimed to be useful in the treatment of diabetes (Personal information from Villagers of Anagipuram, Suryapet district, Telangana, India). The present study was conducted to verify the claim and evaluate thein vivo antihyperglycemic property of the roots of D. cinerea by preparing the aqueous alcoholic extract followed by its partitioning with various organic solvents, and the resultant extracts tested for glucose tolerance test to provide the scientific evidence. High-performance thin-layer chromatography (HPTLC) analysis and anatomical (transverse section) characters were also investigated.

  Materials and Methods Top

Collection of plant material

The fresh roots of D. cinerea were collected from Suryapet, Telangana, India. The plant authentication was done in Botanical Survey of India, Hyderabad. The voucher specimen (GPRCP/DC/BR07/2015) was maintained in the Department of Pharmacognosy, G. Pulla Reddy College of Pharmacy, Hyderabad. Fresh roots were used for anatomical studies, shade-dried roots were used for HPTLC and pharmacological studies.

Microscopical studies

The free-hand transverse sections were first treated with chloral hydrate reagent, mounted in glycerin, and observed for histological characters using compound microscope. The presence of starch grains was identified by treating the section with dilute iodine solution and the presence of lignified tissues such as xylem and phloem were determined by treating the section with phloroglucinol and concentrated hydrochloric acid (1:1).[12],[13]

Preparation of ethanolic extract

The shade-dried roots powder (#60) was extracted with 80% ethyl alcohol by maceration for 8 days. The percentage yield of aqueous ethanolic extract is 5%.[14]

Fractionation of the mother extract

To the 100 g of concentrated ethanolic extract, 500 ml of distilled water were added and fractioned with petroleum ether (4 × 500 ml), chloroform (4 × 500 ml), ethyl acetate (4 × 500 ml), and butanol (4 × 500 ml). The percentage yields of fractions are 1.8%, 4%, 12%, and 30%, respectively.[15]

Test animals

Male Wistar rats (~3 months age, weighing 180–200 g) were procured from the National Institute of Nutrition, Hyderabad, India. Animals were maintained in cages under standard environmental conditions of temperature, relative humidity, dark/light cycles, and free access to feed and water ad libitum during the quarantine period. Animals were fasted ~16 h before experiment but had been allowed free access to water. The Institutional Animal Ethics Committee of G. Pulla Reddy College of Pharmacy, Hyderabad, India, has approved the animal experimental protocols (GPRCP/IAEC/02/14/8/PCG/AE-1-RATS-M188-mice-m-30). All the extracts of D. cinerea root were tested for glucose tolerance test at doses of 200 and 400 mg/kg and were administered orally as fine aqueous suspension of 0.5% w/v carboxymethyl cellulose (CMC) as vehicle.

Toxicity studies

Acute toxicity studies were performed according to the Organisation for Economic Co-operation and Development 425. The test uses a maximum of five animals. A test dose of 2000 mg/kg was used. The first animal was dosed and observed continuously for the initial period of 2 h, intermittently for next 6 h, and then 24 h for death and abnormality in behavioral changes, the animal survived then next four animals dosed sequentially. All the animals survived-LD50 was >2000 mg/kg.[16]

Effect of Dichrostachys cinerea root extracts on glucose tolerance in rats

Overnight fasted rats were divided into 12 groups of 6 rats each. Group 1 served as control, received vehicle. Group 2–11 received various CMC suspensions of D. cinerea extracts at an oral dose of 200 and 400 mg/kg. Group 12 received glibenclamide as standard at an oral dose of 10 mg/kg. After 30 min of extract administration, the rats of all groups were orally loaded with 2 g/kg of glucose. Blood samples were collected from the retro-orbital plexus just prior to glucose administration and at 30, 60, and 90 min after glucose loading. Plasma was separated, and blood glucose levels were measured immediately by glucose-oxidase method.[17],[18],[19]

The percentage variation of glycemia was calculated for each group using the formula:

Percent variation in glycemia = Gi − Gt/Gi × 100


Gi – Value of initial glycemia (0 h) and

Gt – Glycemia at 30, 60, and 90 min, respectively.

Statistical analysis

All the values were expressed as mean ± standard error of the mean, results were analyzed statistically using analysis of variance followed by Dunnett's test. Values of P < 0.05 were considered statistically significant.

High-performance thin-layer chromatography analysis

The all extracts of D. cinerea were dissolved in HPLC grade methanol (100 mg/1 ml). The solution was centrifuged at 3000 rpm/5 min and used for HPTLC analysis. Two microliters of the samples were loaded as 8 mm band length in the 5 × 10 precoated silica gel 60F254 aluminum-supported TLC plate using Hamilton syringe and CAMAG Automatic TLC sampler (ATS4) instrument.

HPTLC plates of all fractions were developed (up to 90 mm) in the twin-trough chamber with the respective mobile phase. After developing, the individual plate was kept in the photodocumentation chamber (CAMAG TLC Scanner-180114) and captured the images with WIN CATS software (version 1.4.6), Muttenz, Switzerland at ultraviolet (UV) 366 and 254 nm. Ethyl acetate and butanol fraction TLC plates were derivatized with phenolic spraying reagent (20% of sodium carbonate followed by spraying of Folin–Ciocalteu [FC] reagent).[20],[21],[22]

  Results Top

Morphological charters

D. cinerea root pieces are straight, cylindrical, woody, exhibiting a longitudinal ridge and narrow furrow, distinct nodes, abundant fibrous roots at a distance. The root bark is thin, yellow-to-buff color and having brown patches externally, whitish gray internally, odor characteristic and taste bitter. Fracture-very hard to break, uneven and fibrous.

Microscopical characters

Root shows circular outline, consisting of distinct secondary characters, the following tissues were observed from periphery to center.

Cork – Multilayered tangentially elongated isodiametric suberized cells, dark gray in color, and few starch grains are present [Figure 1].
Figure 1: Transverse section of Dichrostachys cinerea root a - Cork; b - Phelloderm; c - Resinous matter; d - Cortex; e - Starch grains; f - Phloem fibers; g - Phloem; h - Xylem; i - Medullary rays

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Phelloderm – 2–3 layers, consisting of thick-walled polygonal parenchymatous cells and consisting of yellowish-brown resinous matter.

Cortex – Many layers of thin-walled cellulosic parenchymatous cells containing numerous starch grains.

Phloem – Phloem is wide, thin-walled cells, consisting of numerous lignified phloem fibers.

Secondary xylem – It occupies major part of the roots and is composed of lignified xylem vessels, xylem parenchyma, and thick-walled cells. Xylem vessels appear rounded or polygonal and occur in single. The central wood is excentric.

Medullary rays – The medullary rays run radially from center to the cortex through the phloem. They are uniseriate, lignified, radially elongated, and thick walled. Medullary rays in the cortex region are wider and lignified.

Toxicity studies

In oral acute toxicity studies, no mortality and abnormal behavioral changes observed in mice up to a dose of 2 g/kg body weight. All extracts of D. cinerea was found to be safe, and further antihyperglycemic activity was tested at an oral dose of 200 and 400 mg/kg body weight.

Effect of Dichrostachys cinerea root extracts on glucose tolerance in rats

The effect of D. cinerea extracts in glucose tolerance test is given in [Table 1]. The maximum glucose concentration of diabetic control is 179.02 mg/dl at 60 min. After glucose load, the raise in glucose concentration is observed from 30 min and reached maximum at 60 min and decreased by 90 min. All extracts have produced significant dose-dependent activity; however, the maximum significant (P < 0.01) activity was produced at 400 mg/kg by ethyl acetate extract, followed by ethanol and butanol extract with percent protection of 52.32, 40.34, and 35.24, respectively, at 60 min of experiment. The standard glibenclamide (10 mg/kg) produced a significant maximum protection at 60 min (64.24%) and none of the extracts activity is comparable with standard activity at time of experiment. Leftover aqueous extracts not produced any hypoglycemic properties.
Table 1: Effect of Dichrostachys cinerea root extracts on glucose tolerance test in rat

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High-performance thin-layer chromatography analysis

HPTLC analysis of ethanolic extract showed the presence of seven constituents at different Rf values at 366 nm. Similarly, chloroform fraction contained 10 constituents (366 nm), ethyl acetate fraction 10 constituents (254 nm), butanol fraction 7 constituents (366 nm), and aqueous fraction 7 constituents (366 nm). When all plates were observed at UV-254-nm fluorescence quenching was observed, which was seen as dark blue zones on the green background of the TLC plates. After derivatization with the respective derivatizing agent (FC reagent) on the ethyl acetate and butanol fraction TLC plates, dark blue color spots were observed which indicates the presence of phenolic compounds. The results are summarized in [Table 2] and [Figure 2], [Figure 3], [Figure 4].
Table 2: Mobile phase and Rf values of different fractions

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Figure 2: Scanned and derivatized high-performance thin-layer chromatography plates

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Figure 3: Developed high-performance thin-layer chromatography chromatograms of ethanolic extract and chloroform fraction

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Figure 4: Developed high-performance thin-layer chromatography chromatograms of ethyl acetate and butanol fraction

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  Discussion Top

D. cinerea root decoction is claimed to useful in diabetes. Results of hypoglycemic activity of D. cinerea root extracts presented here may help to establish a scientific basis for the utility of this plant in the treatment of diabetes. The findings of this study have demonstrated that the ethyl acetate and aqueous ethanolic extract produced maximum antihyperglycemic activity. The efficacy of the ethyl acetate and aqueous ethanolic extract dose is higher than the hypoglycemic activity of glibenclamide (10 mg/kg) in the glucose loaded diabetic rats. Glucose tolerance test is a primary testing method to access efficacy of test substance. The oral route of administration was used, as it is simple and physiological. Male Wistar rats were chosen for the experiment because the blood glucose levels of rats remain stable during handling, unlike other animals such as rabbits. Derivatized HPTLC plate of ethyl acetate extract revealed the presence of phenolic compounds, which may contribute to the hypoglycemic activity. As far as the mechanism of action is concerned, we can speculate that the hypoglycemic activity of D. cinerea could be due to an enhancement of peripheral metabolism of glucose, even if an increase insulin release cannot be excluded.

Anatomy of fresh roots revealed the presence of multilayered cork cells, yellowish-brown resinous matter, lignified phloem fiber, and xylem vessels. The HPTLC analysis of mother extract and all other fractions showed the presence of different constituents at different Rf values at 366 nm and 254 nm (ethyl acetate fraction). HPTLC chromatograms obtained in the range of 250–400 nm shown clear peaks for all fractions. These anatomical and HPTLC studies will be helpful in identifying the drug either in entire form or in powder form.

  Conclusion Top

The transverse section of D. cinerea roots shown several diagnostic characters such as isodiametric suberized cells, parenchymatous cells containing numerous starch grains, and lignified unitriseriate elongated medullary rays. HPTLC chromatograms of ethyl acetate and chloroform fractions shown ten peaks, hydroalcoholic and butanol fractions shown seven peaks, which help's as a fingerprint for identification and standardization. This anatomical and chromatographic analysis can be considered as reference standards for future studies on D. cinerea roots. Ethyl acetate and hydroalcoholic extracts showed significant antihyperglycemic activity at 400 mg/kg dose among all extracts. Presence of phenolic compounds in ethyl acetate fraction may be responsible for its antihyperglycemic activity. Further, D. cinerea roots deserve extensive research to isolate active components responsible for antidiabetic activity.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2]


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