Calophyllum Inophyllum Linn Is One of the Important Ayurvcdic Drug Plants of Which Practically Au Parts Are Used Medicinally

Proc. Indian Ac-ad. Sci. (Plant Sci.), Vol. 93, No. 6, Novcmber 1984, pp. 643-654 Printed in India.

Pharmacognostic studies on the flower of Calophyllum Ÿ Linn

SHANTA MEHROTRA, USHA SHOME and H P SHARMA Pharmacognosy Laboratory. National Botanical Research lnstitute, Lucknow 226001, India

MS reccived 24 May 1983; reviscd 13 June 1984

Abstract. Calophyllum inophyllum Linn is one of the important Ayurvcdic drug plants of which practically aU parts are used medicinally. Among thesc flowers and stamens are oftcn uscd asa substitute for 'Nagkesara'. This paper deals with a detailed pharmacognosy of the flower of C. inophyllurn. Physico-chemical constants, preliminary phytochemical studics. fluorescence analysis, behaviour of powdcred drug with different chemieal reagents and thin layer chromatography were also carricd out.

Keywords. Calophyllum inophyllum; flower; pharmacognosy; Cluseaceae.

I. lntroduction

Calophyllum inophyllum Linn (family-Cluseaceae) which is variously known as Undi (Hindi); Sultan champha (Bengali); Nagchampa, Nagkesara (Sanskrit) and Alexender Laurel (English) is an important Ayurvedic drug plant and is used asa substitute for 'Nagkesara" (Chunekar 1960; Shaligram 1953; Singh and Chunekar 1972). Different parts of this plant are variously valued for their digestive, antipyretic, diaphoretic and antidysentric properties. The decoction of the flowers taken intemally, is said to cure syphilitic eczema and insanity (Rao 1914). Calophyllum inophyllum is an ornamental tree with a thick trunk and smooth grey bark, large smooth shining leaves with white sweet scented flowers, occumng along the coastal regions of the southern India, Andaman lslands, Burma and Ceylon. A good amount ofwork on the chemistry of the seed oil, bark, heart wood and leaves of C. inophyllum has been carried out by va¡ workers. Subramanium and Nair (1965, 1971) isolated myricetin-7-glucoside from the androecium of the flower, the structure of which was elucidated by Kasim et al (1974). A fair amount ofwork has also been carried out on several botanical aspects of the plant. The morphology and ontogeny of the foliar venation was studied by Ramji (1967), while Gupta and Kundu (1967) studied the parallel foliar venation. Although pharmacognosy of the stem bark was studied by Rai Chaudhuri (1965) that of the ttower has not been carried out so far and hence the present studies.

2. Material and methods

Fresh and dried mate¡ was collected from North Kanara (Karnataka). The voucher herbarium specimen is preserved in the Medicinal Plant Herba¡ at the Central Drug Research Institute, Lucknow (voucher No. 2044). Hand and microtome sections

NBRI Research Publication No. 237 (new ~ries) 643 644 Shanta Mehrotra, Usha Shome and H P Sharma stained with safranin and fast green were used for histological study. For surface study petals and sepals were treated with a dilute solution of nitric acid followed by clearing in chloral hydrate and lactic acid. Physicochemical studies were perforrned with shade-dried material.

3. Gross morphology of the flower

The flowers are fragrant; white; pedicellate; pedicel L-2 cm, slender; occur in axillary solitary of fasicled racemes (figure 1). Sepals 4, reflexed, two outer ones concave (figure 6), inner petaloid. Petals usually 4, imbricate and depressed-globose (figure 24). Stamens indefinite, free or connate al base, filaments small, anthers oblong; ovary superior, unicarpellary, syncarpous, style short, slender; stigma capitate (figures 43--45), ovule single, erect. A young fruit is green to yellow but the mature fruit is brown in colour, globose, tip shortly acuminate and seed ovoi6 or globose.

4. Microscopic characters

4.1 Pedicel A rs of the pedicel is squarish or round to oval in shape (figures 2 and 3) and has a single-layered epidermis with a fairly thick crenate cuticle. The outer layers of the cortex ate collenchymatous, their number being greatly variable (figures 4 and 5). Schizogenous resin canals and prismatic crystals of calcium oxalate are also present in the cortex. The vascular cylinder is ah amphiphloic siphonostele with xylem arranged in groups (figures 2 and 3). The xylem is endarch and comprises of tracheids, vessels, fibres and xylem parenchyma. The phloem is weU-developed and consists of sieve tubes, companion cells and phloem parenchyma. Most of these cells are filled with yellowish brown pigments (figures 4 and 5). The pith is parenchymatous and has schizogenous resin canals (figures 2-5).

4.2 Calyx The epidermal cells of the adaxial surface of the outer sepals are straight and thin walled (figure 7) and devoid of stomata. However, those of the inner sepals ate thick- walled and pitted (figure 8). Both glandular and non-glandular hairs are present on the margin of the adaxial epidc ,'~rª (figures 11-18). The former are 2-3 celled, with slightly elongated base (¡ 11 and 12) whereas the non-glandular ones are usually unicellular sometimes up to 5--celled with pointed or rounded apices (figures 13-18). Epidermal celIs on the abaxial surface are rectangular and have sunken, paracytic stomata with thick ledges (figure 9). Sometimes large twin stomata are also observed (figure 10). Stomatal frequency is higher in the outer sepals as compared to the inner ones. In rs (figures 19-23) the sepal shows a single layer of epidermis covered externaIIy by a thick cuticle. The cells of the upper epidermis are broader than long and devoid of stomata while the lower epidermal celts ate interspersed by sunken stomata. The ground tissue is many-layered and parenchymatous. Pharmacoonosy of C. inophyllum 645

-.-.

:,~-,89162 S ~~,,.~" .,,

c! "

t .'\

9 ". ,e , ~o rnTn

Figure 1. A twig of C. inophyllum with ttower buds.

The vascular bundles are arranged in the middle of the ground tissue. These ate usually amphicribral in which phloem is well-developed while the xylem is represented by 1 ~,~ groups ofvessels (figures 20 and 22). Resin canals ofvarying diameter lined by a layer-of narrow epithelial cells and rosette crystals of calcium oxalate are present throughout the ground tissue (figures 20-22).

P-9 646 Shanta Mehrotra, Usha Shome and H P Sharma

' EP~.....eOL V ~\\\\~

] \\

,~,:~ cc '-,','~:-~--91 .~ ~--'~-~~"~

IFiI~(~ 2-18. 2 i~i 3. rs of pedicel (diagrammatic). 4 znd 5. "rs of pedicel showing details. 6. Sepal showing vasr 7. Epidermal cells of adaxial surfar of outer scpal. 8. Thickwalledepidermalcellsofadaxialsurfaceofinnersepal. 9, Epidermalcellsofabaxial surface ofsepal showing stomata. 10. Epidermal cells ofabaxial surface of sepal showing twin stomata. ! I-12. Glandular hairs. 13--18. Non glandular hairs.

In a rs through the margin the cells are parenchymatous, compact and horizontally elongated (figure 23),

,S.3 Corolla The adaxial epidermis of the outer petals (figure 25) is similar to those of the inner Pharmacognosy of C. inophyllum 647 sepals. However, adaxial epidermal cells of the inner petals are uniform, hexagonal, thin-walled and devoid of stomata (figure 26). There are no glandular hairs on the petals but non-glandular ones, which are uni-celled, with slightly bulging bases and rounded apices are present along the margins (figure 27). Epidermal cells on the abaxial side are variable and have sunken, paracytic stomata like the sepals (figure 28). However, the frequency, and size of stomata are much smaller here and the ledges are thin-walled.

..~~ __~ A~..~-- )9 vs ~,c

ST ~~z L Ep -.."3"~ ~=====~,=~L=,91 23 M

~: /:.\ ~ ': \L

CU___..,,...~/UEP ~-~ 27

--PAR XY

31 2O. Figm'es 19-32 19. TS of sepal (diagrammatic). 20-22. Portions from figure )9 (magni- fied). 23. TS through margin of sepal. 24. Petal showing vasculature. 25. Epidermal cells of adaxial surface of outer petal. 26. Epidermal celts ofadax]al surface of,;nner pe:al. 27. Non glandular hair of petal. 7,8. Epidermal cells of abaxial surface showing stomata. 29. TS of petal (diagrammatic). 30-32. Portions from figure 29 (magntfied).

P-tO 648 Shanta Mehrotra, Usina Shome and H P Sharma

The Ts of the petal is irregular in shape (figure 29). The ground tissue is parenchymatous and is 2-3 layers thick at margins (figure 30) and formed of several layers in the middle (figure 31). The vascular bundles are present in a row and distribution of the xylem and phloem is irregular (figures 29, 31 and 32). As in sepais schizogenous resin canals are distributed throughout the ground tissue (figure 29).

4.4 Androecium The numerous stamens have short thick filaments and elongated anther lobes (figures 33 and 34) as in Mesuaferrea (Shome et al 1982). The stamens are fused at the base and separate upwards (figures 35 and 36). Their staminal bundles ate amphicribral (figure 37). A semi-diagmmmatic representation of the rs of anther lobes is shown in figure 38. The surface cells of the anther lobes ate thick-walled and show close and fine striations (figure 39).

4.5 Pollen grain Pollen grains ate 2-(3-) colporate (average size 34.62• (range 30-47-38.78x27.77-36-01); shape subolate-prolate, spheroidal; exine surface reticulate; thickness !-2 pro, muriduplibaculate, colpus measuring 14 • 38"78/~m (figures 40 -42).

4.6 Gynoeciura The ovary is superior and monocarpellary (figure 46). It has a single-layered epidermis and many-layered ground tissue. The latter is differentiated into an outer 4-8 layered collenchymatous region filled with dark brown pigments and ah inner several-layered parenchymatous region. Schizogenous resin canals are present all over. The ovule is submarginal and anatropous (figures 46 and 47). A rs passing through the style shows a stylar canal in the centre and four resin canals all round (figure 48). The epidermis is single-layered and has a thick cuticle. The ground tissue is parenchymatous and the vascular bundle is amphicribral (figure 49).

5. Powder study

The powder is brown, pleasant smelling and slightly astringent in taste. On microscopic examination the drug powder revealed the following elements (figure 50 A-L): (i) pieces of trichomes (figures 50 A-E), (ii) pieces of thin-walled adaxial epidermis (.figure 50 F) which can be attributed to sepals, (iii) abaxial epidermis with stomata (figure 50 G), (ir) pitted epidermal cells (figure 50 H) probably from inner sepals or outer petals, (v) numerous pollen grains (figures 50 I-K), (vi) rosette crystals of calcium oxalate (figure 50 L). The behaviour of the powdered drug with different chemical reagents was also studied and is presented in rabie 1. The fluorescence analysis carried out according to the methods described by Chase and Pratt (1949) and Kokoski et al (1958) is recorded in table 2. Pharmacognosy of C. inophyllum 649

ii .. ~~_ ~~"~~ C i'. / ii :~ o C

, VS 319, "~"=="~ "

Flll 34

0 - ." , ~ ,~sm, 9 . ~11~.# " 9 ,,~

t4~,-., i t,,~ ~'~~'tf,'. 9 ,,., c

9ltl~~<,,o~ -,-'-,/~ co ~ G u o

0 <'><'- o. o o- <>o'. C o o 0 " o 0 ir ' "w/ ,4~ 50 J K

Figures 33-50. 33. A stamen showing two anther Iobes with fused ¡ 34. A stamen showing one anther lobo. 35. Ts of fusr stamens with vascular supply (dia~ammatic). 391~ of frer stamen (diagrammalic). 3"/. A portion from figure 35 magnifir to show amphicribral vascular bundle, 38. Semi-di91 represr252 of the Ts of anther lobos. 39. The surface cells of anlher lobos showing finr striations. 40. A pollen grain showing wall stratification and ornamr 41. A palynogram of pollen. 42. LO pattern of pollen omamentation. 43-45. Gynor showing stylr and stigma. 46. Ts of ovary showingovu]r252 47. t.sofovary showingsub-marginal ovule (diagrammatic). 48. TS of stylr showing styiar cavity and rr canals (diagrammatic). 49, A por~ion from figura 48 showing details. 5~A-L. Different tissur from the drug powder (for details refer to the text). 650 Shanta Mehrotra, Usha Shome and H P Sharma

Ttble 1. Behaviour of powder on treatment with different chemical reagents.

Powder + Observationz

Powder as such Light brown Picric acid Brown Acetic acid Dark brown Conc. HCI Blackish brown Conc. HaSO~ Light brown lodine (5 ~~ Dark brown Ferric chlo¡ (5 %) Greenish black Sudan III Red Conc. HNO3 + ammonia Orange with black tingr

Table 2. Fluorescence analysis of the powdered drug.

Colour in ordinary Fluorescence Powder + diffused tight under t~v-light

Drug as such Grcyish brown Brown Nitrocellulose in amylacr Dark brown Dark brown IN HCI Brown Greenish brown 1N HCI + nitrocellulose in amylacetate Brown Brown IN NaOH (aqueous) Dark brown Blackish brown IN NaOH (aqueous) + Blackishbrown Brightgreenish nitror in brown amylacetate IN NaOH in rnethanol Blackishbrown Greenish brown IN NaOH in methanol + nitrocellulose in amylacetate Blackish brown Greenish brown 50 % Nitric acid Dark brown Dark purple 50 % Sulphuric acid Brown Greenish brown

6. Phytochemicai studies

Air-dried material was used for quantitative determination of ash values, tannins, sugars and various extractives. The procedures recommended by Anonymous (1966) were followed for calculating total ash, acid insoluble ash, alcohol and water soluble extractive percentages whereas for calculating tannins and sugars Fohlin-Denis reagent and phenol-sulphuric acid methods prescribed by AOAC (Anonymous 1965; Dubois et al 1956) were followed. The results are presented in table 3. Pharmacognosy of C, inophyllum 651

TIble 3. Detcrmination of physico- chcmical constants on air dry wcight basis.

Constants Percentage

Ash Total 2.9389 Acid insoluble 0.169 Tannins 10.6 Sugars (rng/g dry wt) Water soluble 53 Acid insoluble 67 Extractives Alcohol soluble 17 Water soluble 18

Table 4. Phytochemical tests in C, inophyllum Linn.

Extractives

Parameters Hexane Benzene Chloroform Ethanol Aqueous

Total pr by weight 3.883 % 1.585 % 0.747 % 10-974 % 11.759 % Physical Greenish Greenish Greenish Dark brown, Dark brown appearance and brown, waxy, brown, brittle, brown, waxy, tachy, sugary brittle eonsistency very pleasant pleasant smeU pleasant smell smell rcsembling smell Drakshasava Alkaloids - - + + - Flavonoids - - - + + + + + Reducin 8 sugars - - - + + + + + + Resins + + + + - - Saponins .... + Steroids + + + + - - - Triterpr ..... Tannins - - + + + + + +

Total percentage of Extractive = 28.948 % - absent; +_ doubtful; + + appreciable amounts; + + + strongly indicated.

A known quantity of dried material was extracted in a Soxhlet apparatus with hexane, benzene, chloroform, alcohol and water successively. Solvents from the respective extractives were evaporated and percentage of each solid extractive calculated. These were further screened (Peach and Tracy 1955) for steroids and triterperaoids (LB test), flavonoids (Shinoda's test), alkaloids (Mayer's reagent), tannins (ferric chloride test) and sugars (Fehling solution test) and the results are presented in table 4. The respective extractives were further subjected to thin layer chromatography. 652 Shanta Mehrotra, Usha Shome and H P Sharma

6.1 Hexane extractive As many as 11 spots were discernible in unidirectional thin layer chromatography using hexane'benzene (30:70) as the solvent system. Out of these 7 spots were prominent and 4 faint to very faint (figure 51).

6.2 Benzene extractive Using benzene : chloroform (75 : 25) as the solvent system, benzene extractive gave 10 spots. Excepting one spot near the solvent front, the others were quite prominent. Out of these one spot nearer the starting line is characteristically arrow-shaped and dark purple in colour (figure 52).

o E

,J.

I- UJ LU 0 '~ Z ~. ir, LU Z t St HEX~E I[XTgACTIV[ BENas t[ XTRACT~VI[

..... Q b~

9,j y. t ~

O o ,r Di u 6~ 5A CHLOROFOIIM EXTRA91 ALCOHOL [X~,RACT IV~

Figures 51-54. Abbrer¨ 8. brown: BB, blackish brown; DB, dark brown: DP. dark purplc; F, faJnt; FB, faint brown; FY, faint yr LB, [ight brown; LP, light purple; L Y, light ycllow; O, orange; P, purple: PB, purplish brown; VF. vc~ faint; Y, ycllow; YB. ye[lowish brown. Pharmacoonosy of C. inophyllum 653

6.3 Chloroform extractive The chloroform extractive resolved into 5 clear spots anda trail near the start with chloroform : methanol:ammonia (85: 14:1) as the solvent system (figure 53).

6.4 Alcohol extractive The alcoholic fraction gave 12 clear spots in the solvent system chloroform : acetone : n-propyl alcohol (12:4: 1) (figure 54). The hRf values of different extractives in various solvent systems are presented in table 5.

7. Discussion

C. inophyllum L belonging to the 'Nagkesara' (Mesuaferrea) family may be called 'Punnaga' and its flower buds are at present being used in place of real 'Nagkesara' (Singh and Chunekar 1972). The genuine 'Nagkesara' is unanimously accepted to be Mesuaferrea Linn (Dymock et al 1890; Satkopan and Thomas 1967; Vaidya 1971; Waring 1868). The pharmacognosy of the genuine drug was carried out earlier (Shome et al 1982). The present study is an attempt to describe its substitute--C, inophyllum Linn and to differentiate it from the genuine one. It has brought out significant differences particularly in certain characters of pedicel, such as (i) absence ofsemilunar thickenings in the cortex (ii) presence of starch grains in the innermost one or two layers again of the cortex (iii) presence of pericyclic fibres and (iv) abundant rosette crystals of Ca-oxalate in the pith. The other characters which can be profitably used to distinguish the two are presence of glandular hairs on non-essential parts of the flower and unicarpeUary ovary. C. inophyllum also differs markedly from Mesuaferrea L in having a tow percentage ofash 2-938, high percentage oftannin 10.6 (ash value and tannin percentage of Mesua ferrea being 9-297 and 4-4 respectively) and presence of steroids and flavonoids which are totally absent in Mesuaferrea Linn. The aforesaid characters may thus be used to differentiate the genuine 'Nagkesara' from its substitute.

Table 5. hRf values in C. inophyllum Linn.

Extractive Solvent system hRf values

Hexane soluble extractive Hexane:benzenr 98, 87, 75.55, 35, 25, 17. 12, (30:70) 8. 4, 1-5 Benzene soluble extractive Benzene : chloroform 97.9. 83.9, 76.3.59-1, 51.6. 38-7. (75:25) 23.7, 17-3, 9.7. 2.7 Cbloroform soluble extraclive Chloroform : methanol: ammonia 96-8, 86'3, 41"1, 33"1, 28"2 (~5: t4: 1) Alcohol soluble extractive Chlorotorm : acetone : n-propyl 96'7,93.5. 89" I. 82"6. 78.8. 75-0, alcohol 44.6, 30.5, 217, 18.5, 10.9.4.3 (120:40: 10) 654 Shanta Mehrotra, Usha Shome and H P Sharma

The authors are thankful to the Scientist-in-charge for facilities. Thanks are due to Messrs M K Tandon and A Jha for technical assistance.

References

Anonymous 1965 Official methods ofanalysis of the AOAC (Washington DC: BenjamŸ Franklin Station) Anonymous 1966 lndian pharmacopoeia 2nd edn (Delhi: Government of India) Chase C R and Pratt R J 1949 Fluorescence of powdered vegetable drugs with particular reference to development of system of identification; J. Aro. Pharm. Assoc. 38 324-331 Chunekar K C 1960 Commentary on Bhavprakash Niohantu of Shri Bhavmishra (Varanasi: The Claoukhambha Sansknt series) Dubois M, Gilles K A, Hamilton J K, Robers P A and Smith F 1956 Calorimet¡ method for determination of sugars and related substances; Anal. Chem. 28 350-356 Dymock W, Warden C J H and Hooper D 1890 (reprinted in 1972) Pharmacooraphia Indica, A history of the principal drugs ofvegetative orioin I (Dehra Dun: Bishan Singh Mahendra Pal Singh) Gupta B and Kundu B C 1967 ParaUel venation in C. inophyllum Linn; Sci. Cult. 33 123-124 Kasim S M, Ner S, Raman P V and Nair A G R. 1974 Structure of Myricetin glucoside from flowers of C. inophyllum; Curr. Sci. 43 476 Kokoski J, Kokoski R and Slama F J 1958 Fluoresc, enee of powdered vegetable drugs under uv radiation; J. Aro. Pharm. Assoc. 47 715 Peach K and Tracy M V 1955 Modera methods ofplant analysis (Heidr Springer Verlag) Vols. 3 and 4 Rai Chaudhuri H N 1965 Pharmacognostic studies on the stem bark of C. inophyllum Linn; Bull. Bot. Soc. Ben#al 19 54-56 Ramji M V 1967 Morphology and ontogeny of foliar vr of C. inophyllum L; Aust. J. Bot. 15 437--443 Rao M R 1914 Flowering plants of Travancore (Trivandrum: Government press) Satkopan S and Thomas P J 1967 The identity ofAyurvedie Market Drugs I-A. Nagkeshara Mesuaferrea L; Nagarjun 10 461 --467 Shaligram Nighamu Bhushanam 1953 Part 7--8 (Bombay: Khemraj Shri KrishnaDas) Shome U, Mehrotra S and Sharma H P 1982 Pharmacognostic studies on the flower of Mesuaferrea L; Proc. Indian Acad. Sci. (Plant Sci.) 91 211-226 Singh B and Chunekar K C 1972 Glossary of vegetable drur in Brhattrayi (Varanasi: Chowkhambha Sanskrit series) Subramanium S S and Nair A G R 1965 Flavonoids of the flowers of C. inophyllum; Bull. Natl. Inst. Sci. India 31 39 Subramanium S S and Nair A G R 1971 My¡ glucoside from the androecium of the flowers of C. inophyllurn; Phytochemistry 10 1979 Vaidya B 1971 Some controversial drugs of lndian medicine II; J. Res. Indian, Med. 6 95-104 Waring E J 1868 Pharmacopoeia of India 1st edn (Lonflon: W H Allet~)

Abbreviations: A, apex; Al, anther lobr C, cell contents; CN, connective; CO, co!pus; COL, collenchyma; COR, cortex; CR, crystal; CU, cuticle; EP, epidermis; EX, exine; FI, filament; FIB, filament base; GT, ground tissue; LEP, lower epidermis; M, margm; O, ovary; O V, ovule; O W, ovary wall: PA R, parenchyma; PG, pollr grain; PH, phloem; PS, pollen sac; RC, resin canal: SC, stylar canal, ST, stomata; STL stigma; STR, striatioias: TR, t¡162 UEP, upper epidermis; I/, vein; I,'B, vascular bundle: I,'S, vascular supply; XY, xylem