Pharmacognostical Evaluation and HPTLC profiling of the root of
Triumfetta rhomboidea Jacq.
S. Alexandar1, Jyothi M Joy1,2*
1Vinayaka Missions College of Pharmacy (Vinayaka Mission’s Research Foundation Deemed to be University), Salem, India.
2Devaki Amma Memorial College of Pharmacy, Malappuram, Kerala.
*Corresponding Author E-mail: jyothimjoympharm@gmail.com
ABSTRACT:
Bala is an important Ayurvedic drug entering into several ayurvedic medical formulations. Triumfetta rhomboidea Jacq. belonging to the family Tiliaceae, also known as Jhinjharita possesses various properties of the drug Bala. This work involves the pharmacognostical characters, phytochemical analysis and HPTLC profiling of the roots extract of T. rhomboidea. The pharamcognostical study comprises of taxonomical characters of taxa, macro and microscopical characters root, histochemical tests and diagnostic characters. The phytochemical analysis consists of determination of physicochemical constants, preliminary organic analysis and HPTLC profile. Phytochemical studies revealed the presence of alkaloids, glycosides, phytosterols, tannins, saponins etc.
KEYWORDS: HPTLC, Pharmacognostical studies, Phytochemical analysis, Tiliaceae, Triumfetta rhomboidea.
INTRODUCTION:
India is presented with an expensive wealth of medicinal plants. The modern development of botanical medicine wants the mix of scientific results of various areas. Bala is a very important Ayurvedic drug getting in many ayurvedic medical formulations. It is used as one of the ingredient in the treatment of diseases like peptic ulcer, diabetes, urinary calculi, mental diseases, epilepsy to mention a few besides giving strength (Balya).The other important property of the drug Bala is that it possesses diuretic activity (Mutrala). Several species of Sida L., two or three species of Abutilon L., two species of Urena L., and one or two species of Pavonia L., and Grewia L., are also mentioned as the botanical sources of Bala in different parts of India. The accepted source of Bala is the roots of Sida cordifolia. Additionally to those sources, Triumfetta rotundifolia Lam., (Tiliaceae), also known as Gippita in ayurveda is reported to be used mutally of the sources of Bala in South India. The allied species T. rhomboidea Jacq.
(Tiliaceae) also known as Jhinjharita possesses some properties of the drug Bala, like Balya (giving strength) 1,2.
Family characters4,5
Annuals or perennial, herbs, shrubs or trees often stellate hairy, mucilaginous. Leaves usually alternate; stipules free, early deciduous; margins simple, entire, sometime lobed, crenate-serrate, mostly palmately veined. Inflorescence axillary, extra-axillary or terminal, of cymes, umbels, racemes, panicles or fascicles, rarely of solitary flower, bracteate. Flowers mostly bisexual, regular. Sepals usually 5, free, sometimes connate, valavte, accrescent or not; epicalyx occasionally present; petals as many as sepals, imbricate or valvate, often with a basal gland beneath; receptacle often raised into a gynandrophore. Disc usually present. Stamens 10-many, free; anther cells 2, dehiscing longitudinally. Ovary superior, 1 to 10-locular; ovules 2-many, axile; style single, divided or 0; stigma entire or lobed. Fruit capsular or drupaceous.
Genus characters:4
Annual or perennial herbs or undershrub. Leaves simple, entire, sometimes 3 to 4-lobed, crenate-serrate. Inflorescence leaf-opposed or axillary, of clusters, cymes, sometimes in terminal racemes. Sepals 5, free; petals 5, glandular below. Stamens 10 to 25. Ovary 1 to 3 locular; style simple; stigma capitate. Capsule globose, covered by setae or tubercles.
Botanical description and distribution:3
Figure 1: Triumfetta rhomboidea Jacq. plant
Herbs or undershrubs. Lower leaves lobed, 4.5 to 7 x 4.5 to 7.2cm, cordate; lobes 3 to 5, obtuse, stellate-pubescent, crenate; petiole 3.5 to 7cm long; upper leaves 2 to 2.5 x 0.6 to 0.8cm, narrowly lanceolate, acute at apex, crenate at base, serrate, stellate-pubescent; petiole short. Flowers yellow, in terminal and leaf-opposed cymes; pedicels up to 1mm long. Calyx-lobes 5, oblong, often shortly apiculate. Corolla lobes, ovate-oblong, glandular and ciliate at base. Torus stamens 10; filaments. Ovary 4 to 5-locular; locules 2-ovuled; style. Capsule, globose, bristly, stellate-tomentose. (Figure 1) T.rhomboidea Jacq. is found throughout tropical and subtropical regions including South India and ascending to an altitude of 1200 m in the Himalayas.
Parts used and Uses:1
Root: Diuretic, Leaves and bark: In dysentery and diarrhea, Fruits, flowers, leaves: Promote parturition when it is delayed, demulcent and astringent. A hot infusion is given to facilitate child birth.
Therapeutic properties and formulations:1,2
T. rhomboidea Jacq. is often used in South India as a substitute of T. rotundifolia. The most important formulation is Kshirabala used in the treatment of rheumatism, mental diseases and nervous disorders to mention a few. Other important preparations where Bala is used as one of the ingredients are: Kumaryasava used in the treatment of diseases like agnimandya (anorexia); parinamasoola (peptic ulcer); prameha (diabetes); asmari (urinary calculi); Cyavanaprasa used in dyspnoea, cardiac diseases, as a rejuvenator; Triphaladi taila used in pratisyaya (rhinitis), kesasarita (hair fall); Manasamitra vataka used in manodosa (mental diseases), unmada (schizophrenia), apasmara (epilepsy), mandabuddhitra (mental retardation); Balarista used in daurbalya (general weakness), agnimandya (anorexia) and vataja roga (rheumatic diseases).
MATERIALS AND METHODS:
Plant material:
The plant material was collected before rainy season from Nagercoil surroundings, situated in Kanyakumari district of Tamilnadu. Identification and authentication of plant material was done by Dr. S. N. Yoganarasimhan, Taxonomist and Research co-ordinator. Taxonomic identification was performed following Gamble (2005)4, Keshavamurthy and Yoganarasimhan (1990)6. Specimen was given a voucher’s collection number and sample of crude drug material was additionally preserved in crude drug deposit.
Pharmacognostical studies:
A small amount of fresh roots were collected and preserved in tubes containing 2% formalin for pharmacognostical work. Macroscopic observations were carried out as per Wallis (1985)7. Microscopical investigations, histochemical tests, macerate and powder analysis was applied following Trease and Evans (2002)8, WHO guidelines (1998)9.
For microscopical studies free hand sections of roots were taken employing a sharp razor. Sections were cleared by warming with a number of drops of chloral hydrate, stained with phloroglucinol: Con. HCl (1:1) then stained with iodine solution and safranin. Sections were then mounted in glycerin with cover slip for microscopical observations10.
Photographs of the images:
Photographs of the images were captured by observing the material under a compound binocular microscope (Olympus – CH 20 I model) with CMOF analogue camera, AV-digitalizer. Special software (Grand VCD 2000+) was used for capturing the images. The images were transferred to a CD after selection and photo prints taken on Kodak paper.
Physicochemical constants:10,11
Physical constants were determined following the standard procedures given in Kokate (1999)13 and Indian Pharmacopoeia (1996)14. It includes moisture content, ash value and extractive values.
Preliminary organic analysis:15,16
Plant extracts were prepared successively by Soxhlation and Maceration. Phytochemical analysis of plant extracts was administrated following Kokate (1999), Harborne (1998), Raaman (2006) and Brain and Turner (1975).
HPTLC studies:
In the present work Camag HPTLC system is used. It is equipped with Linomat V applicator, TLC scanner 3, Reprostar 3 with 12bit CCD camera for photo documentation and controlled by WinCATS- 4 software. HPLC grade solvents obtained from MERCK were used for all the studies. Precisa XB 12A digital balance was used for weighing.
HPTLC profiles of alcohol extract:17,18
In precoated aluminium TLC plate 6µl of alcohol extract solution was applied as 8mm band using Linomat V applicator with a Hamilton syringe. Applied plate was developed in a Camag twin trough chamber (10X10 cm) containing Toluene: Chloroform: Ethanol (28.5:57:14.5) as mobile phase. The precoated plate was developed for a migration distance of 75 mm. Developed plate was then derivatised with Libermann Burchard’s reagent by dipping in it and dried at 1100C in a hot air oven for 10min19,20,21. It was then scanned under all the 3 wavelengths like 254nm, 366nm and 425nm using Deuterium, Mercury and Tungsten lamps respectively and photo documented using Reprostar.
RESULTS:
Microscopical characters:
Transverse section of the root is circular in outline. It consists of cork, secondary cortex, secondary vascular tissue region. Cork is formed of 4 to 6 layers of thick walled tangentially elongated cells, followed by single bedded thin walled cork cambium. Next to cork cambium lies 2 to 3-layered secondary cortex made up of rectangular, thin walled cells; some cortical cells contain rectangular or druses type of calcium oxalate crystals, some contain mucilage in mucilaginous canals while some others are tanniferous; starch grains which are simple and oval are found either singly or in triads or tetrads in some cortical cells. Next to the secondary cortex lies a broad secondary phloem region; a few small and a few big groups of sclerenchyma arranged in 4 to 5 tangential rows occur in this region, alternating with phloem; smaller groups are found on the periphery; each group is made up 15 to 20 thick walled cells. Medullary rays extend into the secondary phloem region and are found interspersed with groups of sclerenchyma; some ray cells are tanniferous while in other secondary phloem cells mucilage canals are found. Next to the secondary phloem lies 1 to 2-layered vascular cambium, followed by a major portion of secondary xylem forming the wood region. Secondary xylem consists of vessels arranged radially, either solitary or in groups of 2 to 4; it is interspersed with patches of intraxylary parenchyma; some cells of intraxylary parenchyma are tanniferous and a few others contain druses type of crystals; mucilage is found in some vessels. Medullary rays are uni or biseriate in secondary xylem, extending from centre to the secondary phloem where it is multiseriate; some ray cells contain simple starch grains. Pith is absent. (Figure 2)
Macerate:
The following parts were observed in macerate studies. Cork cells which are thick walled, rectangular or polygon, contain mucilage, some are pitted, stone cells with giant lumen, rectangular and pitted, fibers that are very long, parenchyma cells which are thin walled, some of different size and shape, with pointed ends, thick walled, with narrow or broad lumen, septate, some with criss-cross thickenings, some spindle shaped, some forked with equal or unequal arms, some crystalloid and vessels of various size and shape, cylindrical, cup-shaped, drum shaped, narrow, some with terminal or subterminal openings or lateral opening, a few with drawn out ends and with bordered pits. (Figure 3)
Powder studies:
When powder was treated with chloral hydrate and alcoholic potassium hydroxide, following elements were observed. Fragments of groups of cork cells which are thick walled and rectangular, fragments of fibers having pointed ends, with large lumen and crystalloid fibers, fragments of groups of parenchyma cells which are rectangular, thin walled, some with mucilage cells or canals, some with rectangular or druses type of crystals and fragments of bundles of vessels with bordered pits. (Figure 4)
Figure 2: T.S of root.
Figure 3: Macerate studies.
Figure 4: Powder studies
Table 1: Results of Moisture content, Ash value and Extractive value
|
Moisture Content |
Ash Value |
Extractive value (%w/w) |
||||||||||
|
Fresh weight (g) |
Dry weight (g) |
Loss on drying (g) |
Moisture content (%) |
Total ash (%) |
Acid insoluble ash (%) |
Water soluble ash (%) |
Petroleum ether |
Benzene |
Chloroform |
Acetone |
Ethanol 70% v/v |
Water |
|
2.00 |
1.83 |
0.17 |
8.5 |
5.49 |
1.44 |
4.16 |
1.30 |
1.44 |
0.68 |
1.34 |
1.82 |
3.74 |
Physical constants:
Results of moisture content, ash and extractive values are presented in Tables 1.
Phytochemical Analysis:
Extracts were subjected to preliminary phytochemical analysis. Water extract showed the presence of alkaloids, glycosides, phenolic compounds, gums and mucilage. Pytosterol, fixed oil and fats were present in pet ether and benzene extract .Ethanol extract revealed the presence of glycosides, phytosterol, phenolic compounds and saponins.
HPTLC studies:
HPTLC profiles of alcohol extract were performed and the following spots were obtained. The alcohol extract revealed 12 phytoconstituents at Rf 0.22, 0.25, 0.29, 0.33, 0.40, 0.44, 0.48, 0.52, 0.60, 0.76, 0.84, 0.95 (Figure 5) out of these spots at the Rf 0.25, 0.33, 0.40, 0.44, 0.48, 0.52, 0.60, 0.76 are most prominent spots and others are less prominent under 254nm (Figure 5). The extract under 366nm revealed 11 phytoconstituents at Rf 0.16, 0.28, 0.31, 0.40, 0.47, 0.49, 0.53, 0.61, 0.69, 0.83, 0.95 (Figure 5) out of these spots at the Rf 0.16, 0.28, 0.40, 0.47, 0.53, 0.61, 0.69 are pronounced and other spots are least pronounced (Figure 5). The same extract under 425nm revealed 11 phytoconstituents at Rf 0.14, 0.25, 0.29, 0.33, 0.40, 0.48, 0.52, 0.60, 0.67, 0.84, 0.95 (Figure 5) out of these spots at the Rf 0.25, 0.33, 0.40, 0.52, 0.60, 0.67 are pronounced and at the Rf 0.14, 0.29,0.0.67, 0.84, 0.95 are least pronounced.(Figure 5)
254nm
366nm
425nm
Figure 5: Image showing the developed plates and chromatograms
The spot at the Rf 0.25 - pink, 0.40 - blue and 0.48, 0.52 and 0.60 showed grey coloured fluorescence under 254 nm. Under 366nm, the spots at the Rf 0.16 - blue, 0.28 - light blue, 0.40, 0.61 – greenish blue, 0.53 and 0.47- light green and 0.69 showed green coloured fluorescence. Under 425nm, spot at the Rf 0.25 – magenta and 0.33, 0.40, 0.52, 0.60, 0.67 showed brown coloured fluorescence. (Figure 5)
DISCUSSION:
The identification of plant material taxonomically and pharmacognostically is important to provide pharmacognostical standards and to avoid spurious or adulterated drugs. Specific diagnostic characters can be identified by performing detailed botanical and pharmacognostical studies. The diagnostic taxonomical characters like the presence of stellate pubescence on the branchlets, flowers in leaf-opposed cymes, capsule stellate-tomentose help in the identification of plant/drug in the field. Microscopical characters presented in the result part help in evolving pharmacopoeial parameters and also in the identification of drug. The physicochemical and phytochemical analysis helps in formulating pharmacopoeial standards. Phytochemical analysis of root extract represents the presence of are alkaloids, carbohydrates, glycosides, phytosterols, triterpenoids, fixed oils, tannins, saponins, gums and mucilage. Further studies on the isolation and identification of specific chemical constituents can be done only after preliminary organic analysis. HPTLC profiling of alcoholic extract of root showed 14 clearly separated spots which may help in determining the biomarker compound of the drug and also to undertake further isolation and identification of the active principle.
CONCLUSION:
Standardization of drugs and formulations of traditional system of medicine is of utmost importance to assure uniform quality and optimum biological efficacy. Scientific parameters based on taxonomical, pharmacognostical and phytochemical studies are essential in identifying and distinguishing the plant from other species. These parameters along with physiochemical constants not only help in standardization of these drugs but also aid in formulating pharmacopoeial standards of drugs. The exomorphic characters have been found to be useful tools to identify the species taxonomically. The qualitative phytochemical investigation gave valuable information about different phytoconstituents present in various extracts, which help future investigators regarding the selection of particular extract for further investigation. HPTLC profiling of alcoholic extract exhibited number of phytoconstituents which will be a fruitful hope for isolation and characterization of compounds. So Triumfetta rhomboidea root can be utilized as a drug for treating various ailments or as a lead molecule.
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Received on 11.01.2021 Modified on 11.06.2021
Accepted on 05.08.2021 © RJPT All right reserved
Research J. Pharm.and Tech 2022; 15(3):1245-1250.
DOI: 10.52711/0974-360X.2022.00208