Decalepis Hamiltonii Wt

Proc. Indian Acad. Sci., Vol. 87 B, No. 2, February 1978, pp. 37--48, 9Printed in India.

Pharmacognostical studies on the root of Decalepis hamiltonii Wt. and Am., and comparison with Hemidesmus indicus (L.) R. Br.*

R C NAYAR, J K PATTAN SHETTY, Z MARY and S N YOGANARASIMHAN Regional Research Centre (Ay.), Government Central Pharmacy, Bangalore 560 011

MS received 13 May 1977; revised 7 September 1977

Abstract. The roots of Decalepis hamiltonii Wt. and Am. are usedinSouth India as a substitute for the well-known Ayurvedic drug' Sariva' (Hemidesmus indicus (L.) R. Br.) which has well-established therapeutic properties. The pharmacognostical details of the roots of D. hamiltonii with illustrations and its comparison with the roots of H. indicus which may be useful for distinguishing the two crude drugs are presented in this paper.

Keywords. Pharmacognosy; Decalepis hamiltonii; comparison with Hemidesmus indicus.

1. Introduction

Decalepis Wt. and Am. (Asclepiadaceae) is a monotypic genus occurring in pennin- sular India (Santapau and Henry 1973). The roots of D. hamiltonii with a strong aromatic odour and a sweet-sarsaparilla-like taste is considered as an appetiser and blood purifier; the roots consist of about 92% fleshy matter and the rest of woody core; the volatile principle responsible for the aroma and taste of the root is 4-0- methyl-resorcylaldehyde (m.p. 42~ the volatile principle has bacteriostatic and toxic properties and hence the root can be stored unaffected for long periods. The roots contain besides the aldehyde, inositol, saponins, a crystalline resin acid, an amorphous acid, a ketonic substance showing the characteristics partly of sterols and partly of resinols; the sterols consist mainly of stigma sterol and brassica sterol; amyrins and lupeols, both free and as esters are also present (Anonymous 1952). Further, it was gathered (personal communication) that in the Government Central Pharmacy, Bangalore and in some of the leading Ayurvedic pharmacies in Tamil Nadu and Kerala, the roots of this taxon which is considered as ' Sariva Bheda ' (in Ayurveda) is used in place of the roots of Hemidesmus indicus (Sariva) in Ayurvedic preparations like Amritamalaka taila, Drakshadi churna, Shatavari rasayana and Yeshtimadhu taila. It finds popular use for pickles. Due to the remarkable similarity in the therapeutic properties of the roots of D. hamiltonii and H. indicus and the non-availability of the roots of H. indicus in bulk quantities as required by drug manufacturers and physi-

*Paper presented by first author at the 29th Indian Pharmaceutical Congress, held at Waltair, December 1977.

37 P. (B)--3 38 R C Nayar et al cians, the roots of D. hamiltonii are used in South India as a substitute for the roots of H. indicus. Aiyer (1951), Datta and Mukerji (1950) and Prasad and Wahi (1965) have worked out the pharmacognosy of the root and root-stock of H. indicus while the pharmacognosy of Ichnocarpus frutescens (L.) R. Br. and Cryptolepis buchanani Roem. & Schult., which are considered as two other substitutes (Krishna Sariva and Jambu- patra Sariva respectively) of 1t. indicus have been worked out by Wahi et al (197In,b). However, available literature shows that the pharmacognostical studies on the root of D. hamiltonii has not been recorded so far, which necessitated the present investigation; a detailed comparative pharmacognostic account of the root of D. hamiltonii and H. indicus is also provided which will help to distinguish them in crude drug form.

2. Morphology of the plant

D. hamiltonii is a stout climbing shrub with jointed stems. Leaves opposite, simple, broadly ovate, coriaceous with unequal base. Flowers small, white, in branched pedunculate cymes. Fruits a pair of stout follicles, lanceolate, ridged; seeds tipped with a white coma (Gamble 1967 (repr. ed.); Ramaswamy and Razi 1973), (figure 1 A, C)--Makaliberu (Kannada), Makalikizhangu (Tamil). Herbarium specimen examined: S N Y Simhan and R C Nayar 0965, colleeted on 6th December 1976, flowering and fruiting, at about 750 m, from Banneraghatta forests, Bangalore district, Karnataka State and Yoganarasimhan 0056, collected on 7th February 1972, fruiting, at about 880 m, Arakanakadu forests, on the way to Malai Mahadeswara hills, Mysore district, Karnataka State, are deposited in the Herbarium of Regional Research Centre, Bangalore. The authentic and market samples of the crude drug are preserved in the crude drug museum of the centre. The plants climb over shrubs and trees; the roots enter deep into the soil, often traversing the crevices of large rocky boulders, making their collection arduous. Part used: Roots.

3. Materials and methods

Freshly collected roots from forests of Banneraghatta surroundings, Bangalore district, Karnataka State, were fixed in 70 ~o alcohol (Johansen 1940) for microscopical studies. Microchemical studies were done on freshly-cut roots according to Johansen (1940), Kay (1938) and Wallis (1965, 1967). For chemical analysis, deter- ruination of physical constants and fluorescence analysis, the shade dried roots were powdered and sieved through 60 mesh following the Indian Pharmacopoeia (Anony- mous 1966) and Chase and Pratt (1949).

4. Macroscopic characters

The roots are 3 to 4 m or more long, traverse deep inside the soil, _emit_a strong aromatic odour when fresh, gradually diminishing on drying; they are markedly Pharmacognosy of D. hamiltonii 39

Figure 1. D. hamiltonii: (A) Plant; (B) Roots; one cut longitudinally; (C) Fruit. Pharmacognosy of D. hamiltonii 41

Figures 2-7. T. S. of 2. root (diagrammatic); 3. portion of pcriderm; 4. secondary phloem and xylem; 5. wood showing meta- and protoxylem; 6. tangential section of wood showing medullary rays; 7. young root. CAM: cambium; MR: medullary rays; OC: oil cells; P: phellem; PD: phelloderm; PG: phellogen; PH: phloem; PI: pith; PX: protoxylem; S: starch grains; STC: stone cells; T: tracheids; TC: thick walled cells; V: vessels. 42 R C Nayar et al fleshy and cylindrical, 3-6 cm across, brownish outside, very pale yellow inside, the outer surface smooth when fresh, soon becomes wrinkled and longitudinally ridged on drying. The smooth transverse surface shows a thin cork and a hard white wood. Fracture is short and splintery (figure 1B).

5. Microscopic characters

The roots are almost circular in outline in transverse section (figure 2). The phellem consists of 6-10 layers of narrow, rectangular, tangentially elongated cells; the cells get compressed excepting for a few inner layers. The suberised cork cells are polyhedral in surface view and are filled with brownish contents (figure 8a); phellogen is one to two layered. Phelloderm consists of 18 to 20 layers of polyhedral cells. In addition, certain thick-walled cells are also present in the phelloderm region. Secondary phloem consists of sieve tubes, companion cells, and phloem parenchyma and here and there a few stone cells (figures 4 & 8i). The cells of the phloem parenchyma are larger towards the peripheral region becoming smaller inside (figure 4).

Figure 8. Macerate: (a) Cork cells; (b) Fibres; (c) Parenchyma ceils of phloem; (d) Starch grains; (e) Tracheids; (f) Laticiferous cell; (g) Wood parenchyma; (h) Vessels; (i) Stone cells. Pharmacognosy of D. hamiltonii 43

Laticiferous cells are non-articulated, branched (figure 8f) and are present in the phloem. Fibres are absent in the secondary phloem region. Wood consists of 3-4 growth rings, composed of vessels, parenchyma, tracheids, tracheid-fibres and medullary rays; all the elements are lignified. Vessels occur singly or in groups of 2-3. The vessels are usually cylindrical, drum-shaped with transverse or oblique articulations and well-marked perforations and much elongated with numerous simple pits (figures 5 and 8h). The tracheids are long with chisel-like tapering or truncate end walls and have simple pits (figure 8e). Fibres are with small oblique pits and thick-walled, elongated, have narrow lumens and tapering ends, forming peg-like outgrowths in some cases (figure 8b). Wood parenchyma cells are simple pitted and are rectangular (figure 8g). Medullary rays are 1-3 cells wide and 6-10 cells high, uni-to multiseriate, the former being more frequent; cells are oblong to radially elongated and are simple pitted (figure 6). Starch grains are present in the phelloderm, secondary phloem, phloem porenchyma and ray cells. They are simple or compound and spherical or sub-reniform (figure 8e, d). Pith is almost absent and the central space is occupied by protoxylem elements (figure 5). Young roots do not show much difference from the mature ones in the structural details but for the range in number of layers of tissues (figure 7). The measurements of different tissues and cells are provided in table 1.

5.1. Powder analysis

The root powder when treated with different concentrated acids like sulphuric, nitric, and hydrochloric acid did not show any reaction; however with glacial acetic acid it turns brown, with NaOH yellow and black with iodine-potassium-iodide solution.

5.2. Macerate

Macerate consists of portion of cork cells, starch grains, phloem parenchyma cells, xylem fibres, tracheids, wood parenchyma, vessels and stone cells (figure 8 a-i).

Table 1. Measurements of different tissues and cells in microns (t0.

PheUem cells T=66-88--100x 26--33--40 M=40--66--78 x 20--26--42 Phelloderm cells T = 26-- 52 -- 72 • 26 -- 65 M = 40 -- 66- 80 • 27-- 40 -- 66 Vessels T=79--170-- 196 M = 150- 375-- 585 • 50-- 114-- 330 Tracheids T=79-- 170-- 196 M=375--420-- 585 • 22--30 Fibres M=600-- 800--930x 15--17--22 Wood parenchyma M =40-- 66-- 78 x 7-- 26-- 30 Xylem ray cells T=21--24--28 x 14--18--21 Pith cells T=14--21--28 Starch grains T=3--4--6 Vessels per sq. mm 120 -- 200 numbers

T = measurements across; M =measurements of macerate. 44 R C Nayar et al

5.3. Cell contents

Tannin occurs as deep brown granules in the cork, secondary phloem and outer layers of phelloderm; latex and volatile oils in the phloem and phelloderm; starch grains in the phelloderm, secondary phloem and ray cells.

6. Phytochemical studies

6.1. Physical constants

The determination of physical constants by standard methods (Anonymous 1966) are given in table 2.

6.2. Organic analysis

Murti (1941) and Murti and Sheshadri (1941a,b, 1942) isolated from the petroleum ether extract of the root of D. hamiltonff, resinols, sterols, fatty acids and essential oils consisting mostly of p-methoxy salycylaldehyde. The subsequent ethanolic extract yielded saponins, tannins and a resin acid C~H2sO10, m.p. 248 ~ besides a small quantity of inactive inositol, m.p. 220 ~ In the present study, 500 g of powdered roots of D. hamiltonii were successively extracted (hot refluxion) with petroleum ether, chloroform, ethanol and water. Yellow sweet smelling waxy mass of petroleum ether extract answered for steroids and triterpenoids. A colourless crystalline substance, m.p. 73-75 ~ separated from the acetone fractions of the extract. Brown coloured chloroform extract was bitter in taste and answered for steroids and carboxylic acids. The petroleum ether and chloroform extracts were run on thin layer chromotography with petroleum ether: chloroform: benzene (2:1:2) as solvent system which gave 5 spots in each for steroids (figure 9). The dark brown semi-solid mass of ethanolic extracts was bitter to taste and answered for phenols, tannins, carboxylic acids and sugars; the water extract answered likewise. The ethanolic and water extracts were worked out for sugars and tannins.

Table 2. Physical constants (proximate analysis)

% loss on drying at II0 ~ 15.56 of total ash 7-80 of water insoluble ash 6-55 alkalinity with 0.1N HC1 (using phenolphthalein) 1.6 ml % acid insoluble ash 3"6 % crude fibre (Min. Ag. UK and USA process) 13"30 extractive principles: (a) Petroleum ether 40--60 ~ 5.12 (b) Ether 0.48 (c) Benzene 1'30 (d) Chloroform 1.82 (e) Ethanol 12.22 (f) Water 2.14 solubility (a) Water 18.96 (b) Ethanol 16.48 % volatile oil 1.062 Pharmacognosy of D. hamiltonii 45

14-3 3-2(~

12.'

8.8~ osg ~ 7..~ (~ 08., 790 ~ ,.z ~z.5

e.3 s.eD ,~..5 ~ 0 s,

3,o~ 0 3.0 z.4(~ C~2.4

01 02 O O O (> D.H. D.H. H.Z; D.H. D.H. PI.Z. Fig, 9 Fig. 10 Fig. I| ~

Figures 9-11. 9. Chromatogram of D. hamiltonii: 1. Petroleum ether extract; 2. Chloro- form extract. System:--Peteroleum ether: Chloroform: Benzene (2:1 : 2). Developer sulphuric acid. 10. Comparative chromatograms of hydrolysed ethanolic extracts of H. indicus and D. hamiltonfi. 11. Comparative chromatograms of ethanolic extracts of D. hamiltonii and H. indicus. System for 10 & 11: n-butanol: acetic acid: water (5:1 : 4). D.H.:D. hamiltonit; H. I.=H. indicus.

The tannin fraction of both these extracts were submitted to ascending paper chromatography using benzyl alcohol: 1-butanol: 1-propanol: water (6:2:2:2) as solvent system and alcoholic ferric chloride as developer. One spot in each fraction of these extracts was obtained (Rf value 0.72). The sugar fraction was submitted to ascending paper chromatography using isopropanol: pyridine: water: acetic acid (8:8:4:1) solvent system with ammonical silver nitrate as developer. Two brown spots (reducing sugars) of Rf values of 0"62 (sucrose) and 0-57 were developed in each fraction of these extracts.

7. Comparison of D. hamiltonii and H. indicus

D. hamiltonii differs morphologically from H. indieus in being stout climbing shrubs instead of slender prostrate, semi-erect or twining herbs or undershrubs; having leaves which are ovate-lanceolate instead of elliptic-linear; flowers in pedunculate cymes instead of sub-sessile cymes. Macroscopically D. hamiltonii roots differ from H. indicus being dull brown, smooth, stout and straight instead of dull red to dark brown, rough, slender and curved; microscopically in having 6-10 layers of cork ceils instead oF 4--]4 layers, stone cells present in secondary phloem instead of absent, ray cells absent in the phloem instead of present. The chemomicroscopic and cell contents in both were found to be similar. 46 R C Nayar et al

7.1. Fluorescence analysis

The root powders of both the taxa were examined under the same wave length of the ultraviolet light and the results are given in table 3.

Table 3. Fluorescence analysis of the root powders of D. hamiltonii and 11. indicus.

D. hamiltonii H. indicus Treatment visible short wave long wave visible short wave long wave of powder light (254 mr*) (365 into light (254 m/~) (365 mr0

As such pale no fluor- white fluor- golden no fluor- white brown escence rescence yellow rescence fluorescence In methanol brown -do- -do- Grey -do- bluish white In ethanol -do- -do- -do- -do- -do- -do- 0"1 N NaoH Dark -do- greenish- Grey -do- Greenish- in methanol brown yellow yellow 0"IN HC1 Pale -do- pale pale -do- few green brown greenish- yellow specks yellow

7.2. Chromatographic studies

Chloroform extracts of the roots of D. hamiltonii and H. indicus were subjected to thin layer chromatography with n-butanol: acetic acid: water (5:1:4) as solvent system and studied under uv; subsequently the plates were sprayed with basic lead acetate and again studied under uv. The distinguishing characters of the two systems are given in table 4.

Table 4. Comparative chromatographical studies under u.v. on the roots of D. hamiltonii and H. indicus (Solvent system n-butanol: acetic acid: water, 5 : 1 : 4)

Plate without treatment Plate treated with lead acetate D. hamiltonii H. indtcus D. hamiltonii 1-1. indicus Chloroform extract Short wave no spots 2 yellow 2 yellow and 2 yellow spots spots 1 red spot Long wave no spots no spots 1 blue spot 2 blue spots Ethanol extract Short wave 3 brown spots no spots 3 brown spots no spots Long wave 3 brown spots no spots 3 brown spots no spots Hydrolysed fraction Short wave 1 blue spot 1 blue spot 1 blue spot 1 yellowish- blue spot Long v~ave 1 blue spot 1 yellowish- 2 blue spots I yellow blue spot 1 yellow spot spot; 1 blue spot

Similarly ethanolic extracts of both drugs were studied as such and under u.v. these were subsequently hydrolysed (with 4N HC1), neutralised and extracted with ethyl acetate and the results are given in table 4 (figures 10 and 11). Pharmacognosy of D. hamiltonii 47

8. Conclusion

The above study indicates that although D. hamiltonii and H. indicus belong to taxo- nomically different genera, the root (crude drug) of both these taxa exhibit very similar characters or properties in chemomicroscopy and in organic analysis as pointed out by Murti and Sheshadri (1941b). These similar characters coupled with the similar therapeutic properties support the fact that the roots of 1). hamiltonii could be used as a useful substitute for the roots of H. indicus. However, fluorescence analysis and chromatographic studies of the drugs differ which help in distinguishing the two in crude drug form.

Acknowledgements

The authors are grateful to Drs P N V Kurup, K Subramanyan and B M Sharma for facilities and encouragement in this work; to A R Sharma, for assisting in some of the pharmacognostical studies.

References

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