Challenges on Standardizing the Closely Related Herbal Plants of Ocimum Species

MadhumathiSundararajan*1, Vasanth KumarMohan1 and ThangamaniPakkirisamy1

1East West Integrative Medicine Hospital and Research Institute, B-13 Mogappair industrial Estate, Mogappair,Chennai-600037,Tamil Nadu,India.

Author for correspondence:

MadhumathiSundararajan

Email:

Abstract

Plan:Ocimum species are reported to have a great medicinal value in Indian medicine. Leaves of Ocimum sanctum and Ocimum basilicum are commonly known as holy basil and sweet basil respectively belongs to the family lamiaceae. Methodology: Present work is related to differentiate and standardize the Ocimum sanctum and Ocimum basilicum closely related medicinal plants by using powder microscopy and thin layer chromatography (TLC). Outcome: The study revealed the importance of powder microscopy and TLC techniques to identify the closely related species.

Key words: Ocimum species, powder microscopy and TLC

1. Introduction

The Siddha and Ayurveda manufacturing industries and other herbal pharmaceutical industries is facing major problem is identification and quality control of crude herbal drugs1. The proper identification of the drugs has become difficult owing to its different synonyms and vernacular name and different species in same genera.

However, along with the identification of original plants the adulterant crude drug and powder drug should be identified. Nevertheless the light microscopy and thin layer chromatography are the only easy and cost effective method of differentiation of crude drugs.

Among the plants known for medicinal value, the plants of genus Ocimum belonging to family Lamiaceae are very important for their therapeutic potentials. Ocimum sanctum (Holy basil) is an erect herb which is grown every part of the India. Leaves are long in size, broad, elliptical, oblong in shape2several medicinal properties have been attributed to Ocimum sanctum. Different parts of Tulsi plant e.g. leaves, flowers, stem, root, seeds etc. are known to possess therapeutic potentials and have been used, by traditional medical practitioners, as expectorant, analgesic, anticancer, antiasthmatic, antiemetic, diaphoretic, antidiabetic, antifertility, hepatoprotective, hypotensive, hypolipidmicand antistress agents. Tulsi has also been used in treatment of fever, bronchitis, arthritis, convulsions etc.3-8.

Ocimum basilicum (sweet basil) is an aromatic plant, nearly glabrous branching herbs, 60-90cm in height with hairy stems, branches green and widely used in traditional Chinese medicine to treat cardiovascular diseases including hypertension9

The present study was aimed to distinguish the difference between the Ocimum species, further the comparative analysis of TLC has been made to conform the powder microscopy findings.

2. Materials and Methods

2.1. Plant collection

The plant species were collected from the southern state. Fresh leaf materials were procured. The leaves were cleaned, dried in shade and powdered.

2.1.2. Macroscopic evaluation of Ocimum sanctum and Ocimum basilicum

Various macroscopic characters of fresh leaves of Ocimum sanctum andOcimum basilicum were recorded such as duration, type of leaf base, presence or absence of petiole and characters of Lamina. Lamina consists of characteristic features such as composition, incision, shape venation, apex, base surface and texture.

2.1.3. Powder microscopy analysis of Ocimum sanctum and Ocimum basilicum

Finely powdered each raw material was taken and boiled in chloralhydrate separately to remove chlorophyll if present. Then it was stained first with phloroglucinol solution and after few minutes, it was treated with concentrated hydrochloric acid at 1:1 ratio. After 3 minutes of the above treatment, it was mounted with glycerine and observed under microscope. Similarly, the presence of starch grains was also established by staining the powder samples with 1 % iodine solution. Similarly the presence of calcium oxalate crystals was also tested using standard procedure 10.

2.1.4. TLC Derivatization of Ocimum sanctum and Ocimum basilicum

Five gram powder of the each raw material was soaked separately overnight in methanol. Then it was boiled in a water bath to remove methanol. Aluminium TLC plate pre coated with silica gel G/UV 254 of 0.2 mm thickness (Merck) was used for the TLC analysis.

Battery of solvent systems was used to perform thin layer chromatography such as toluene: ethyl acetate (9.3:0.7), dichloromethane: chloroform (2:8), chloroform: methanol (7:3) and n-hexane: ethyl acetate (6:4).

The 20 µl sample was loaded on TLC plate and was allowed to run to cover the 70% distance of the TLC plate. Then the plate was dried and stained with 10 % Sulphuric acid. The resolved bands were identified using standard procedure 11.

3. Results

3.1. Macroscopic evaluation of Ocimum sanctum, Ocimum basilicum

Ocimum sanctum leaves are long and broad in nature. Shapes of the leaves are oblong. Entire margin. Pubescent surface on lower and upper side with minutely gland dotted.

Ocimum basilicum leaves are entire and opposite arrangement. Green or purple in colour and ovate in shape.Toothed margin and glabrous surface (Fig.1).

Ocimum basilicum Ocimum sanctum

Fig.1Morphological view of Ocimum sanctum and Ocimum basilicum

3.2. Powder microscopy analysis of Ocimum sanctum, Ocimum basilicum

3.2.1.Ocimum sanctum

Non glandular covering trichromes are common in powder. They are thick, one cellular head, stalk comprising two upper cells and two lower cells. The body cells have dense cytoplasm and prominent nucleus. Numerous presences of volatile oil components are thick walled, oval in shape and brown in colour comprising schizogenous cells. Diacytic stomata present occasionally.

Vascular elements are not present commonly. Two types of fibres are seen in the powder. Some of the fibres have wide lumen and thin wall and their end tapered abruptly. Second types of fibres are thick walls and narrow lumen. The cells are gradually tapering at the end into pointed tips. The powder also showed the presence of well-arranged annular vessels (Table.1 & Fig.2).

3.2.2.Ocimum basilicum

Isolated capitated type of glandular trichomes and Non glandular covering types of trichomes are seen (Table.1& Fig.2).

S.NO / Parameters / Ocimum sanctum / Ocimum basilicum
1. / Trichomes / Numerous unicellular head, multicellular stalk covering trichomes. / Multicellular trichomes
2. / Glands / Small short with multi cellular stalk / Multi cellular head and multicellular stalk
3 / stomata / Diacytic / Diacytic
4 / Others / Vessels and oil glands / Oil glands

Table.1. Powder microscopy analysis of Ocimum sanctum, Ocimum basilicum

Stomata
/ xylem vessels
Simple trichomes
/ non- glandular trichomes

Fig.2Powder microscopic analysis of Ocimum sanctum, Ocimum basilicum

3.3. TLC Derivatization of Ocimum sanctum, Ocimum basilicum

The resolution of different kind of chemical composition has been separated using TLC and the Rf values were calculated in order to standardize the drug for its identity, purity and strength. TLC finger printing of different extracts of Ocimum species leaf was done and phytoconstituents were separated in eight different mobile phase of varying polarity. Among all chloroform: acetone (9:1) mobile phase was suitable for methanol extracts.

The TLC studies of methanolic extract shows best separation using chloroform: acetone (9:1) as a mobile phase. The chromatogram obtained showed 4 spots on Ocimum sanctum at Rfvalue in white light and 5 spots on Ocimum basilicum at Rf value in normal light. The spots at Rf value was pink under UV radiation (Table. 2 & Fig.3).

S.No / Ocimum sanctum / Ocimum basilicum
1 / 0.36 / 0.31
2 / 0.42 / 0.36
3 / 0.51 / 0.40
4 / 0.65 / 0.44
5 / 0.65

Table.2 TLC Derivatization of Ocimum sanctum, Ocimum basilicum

  1. Ocimum sanctum
  2. Ocimum basilicum

Fig.3 TLC Derivatization of Ocimum sanctum, Ocimum basilicum

3.4. Relative abundance

The greater proportion of the tissues recovered from the samples consisted of non-glandular covering trichomes and glandular trichomes. Whereas thick walled, oval, volatile oil cells were not common and when present. From Table.1 can be seen that Ocimum sactum retained more characters in its powder from then other species. The herbal species show very little resemblance on the basis of these characters as suggested by the prevalence of low resemblance co efficient among them in Table.2.

4. Discussion

According to the WHO , the macroscopic and microscopic description of a plant is the first step to establish the identity and the degree of purity of such materials and should be carried out before any test are undertaken12

The present study investigates the powder microscopy analysis of closely related Ocimum species. Leaves powder of both Ocimum sanctum and Ocimum basilicum were studied in detail to highlighted important histological characters. Ocimum sanctum comprise of Non glandular covering trichromes are common in powder. They are thick, one cellular head, stalk comprising two upper cells and two lower cells. Nevertheless Ocimum basilicum contains glandular and non -glandular trichomes with multi cellular head. Other histological characters like dyacytic stomata, oil glandsand vascular elements were commonly present in both the plants.

The characters available in the powder form of the specimens are not potentially useful for distinguishing the sample in a mixture. The paucity of the characters obtained from the species suggests the inadequacy of histological techniques in detecting the presence of this species in the powder form.

Several herbal products in the market cannot be identified using morphological and histological difference 13.Thin layer chromatography offers an alternative qualitative profile and is being increasingly used for the authentication of crude drugs or their extracts.

The present study investigates the thin layer chromatography analysis of Ocimum species. The TLC photo documentation of the Ocimum sanctum under UV 254 nm shows three major spots at Rf 0.36, 0.42 & 0.65 and Ocimum basilicum under UV 254 shows 4 major spots at Rf 0.31, 0.36, 0.40 0.65 and others are minor spots. The achieved results of thin layer chromatography shown the differentiation on spots Rf values and numbers of spots between the Ocimum sanctum and Ocimum basilicum will be useful tool for authentication, standardisation and quality control assessment of closely related species14.

An examination by microscopy alone cannot always provide complete identification though when used in association with other alternatives techniques. TLC is the most versatile techniques for the identification of related species.

5. Conclusion

The present study was undertaken with a view to lay down standards which could be useful to detect the authenticity of the raw materials. Therefore the use of powder microscopic analysis of herbal medicine is cheaper and more rapid when it is justified along with TLC profiles.

Reference

  1. Yadav Pramod, Harisha CR, Prajapati PK. Validation of pharmacopoeial characters of Bhringaraja (Eclipta alba Linn.Hassk.) Journal of Current Pharmaceutical Research. 2011; 8 (1): 17-24.
  2. Choudhury Golak Bihari, Behera Manaswini, Jena Prabhat Kumar and Tripathy Sujit Kumar. Pharmacognostical and phytochemical investigation of various tulsi plants available in south eastern Odisha. International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701. 2011.
  3. Batta SK, Santhakumari G. The antifertility effect of Ocimum sanctum and Hibiscus rosa sinensis. Indian J Medical Research 1971; 59: 777–781.
  4. Reghunandana R, Sood S, Reghunandana V, Mehta RM, Singh GP. Effect of Ocimum sanctum Linn (Tulsi) extract on testicular function. Indian J Medical Research 1995; 49(4): 83–87.
  5. Nagarajun S, Jain HC, Aulakh GS. Indigenous plants used in the control of Diabetes. In: Cultivation and utilization of medicinal plants. Editors: Atal CK and Kapoor BM (Published by PID CSJR) 1989, p. 584.
  6. Mnadal S, Das DN, Dey K. Ocimum sanctum Linn - A study on gastric ulceration and gastric secretion in rats. Indian J Physiol Pharmacol 1993; 37: 91–92.
  7. Sethi J, Sood S, Seth S, Thakur A. Protective effect of Tulsi (Ocimum sanctum) on lipid peroxidation in stress induced by anemic hypoxia in rabbits. Indian J Physiol Pharmacology 2003; 47(1): 115–119.
  8. Sarkar A, Pandey DN, Pant MC. Changes in the blood lipid profile level after administration of Ocimum sanctum (Tulsi) leaves in the normal albino rabbits. Indian J Physiology Pharmacology 1994; 38(4): 311–312.
  9. AnwarUmar, GuzelnurImam, WuliyaYimin, ParhatKerim, IbadetTohti, BénédicteBerkéand NicholasMoore. Antihypertensive effects ofOcimum basilicumL. (OBL) on blood pressure in renovascular hypertensive rats.Hypertension Research(2010)33,727–730.
  10. Khandelwal KR, Practical Pharmacognosy: Techniques and Experiments, Nirali

Prakashan, Pune; 2002.

  1. Stahl I. Thin layer chromatography, A Laboratory Hand Book (student edition). Springer-Verlag, Berlin; 1969.
  2. Jayeola Adeniyi A. Micromorphological study of plant fragments in some powdered medicinal plants. Journal of Medicinal Plant Research. 2009; 3(5):438-442.
  3. Springfield EP, Eagles PKF, Scott G (Quality assessment of South African Herbal Medicines by means of HPLC fingerprinting. J. Ethnobotany. 2005; 101(1-3): 75-83.

14. Vieira RF, Simon JE. Chemical characterization of basil (Ocimum spp.) found in

the markets and used in traditional medicine in Brazil. Economic Botany.2000; 54:207–216.