|SYMPOSIUM - HERBAL DRUGS AND BOTANICALS - RESEARCH ARTICLES
|Year : 2015 | Volume
| Issue : 4 | Page : 254-259
Quality control aspects of herbs and botanicals in developing countries: Coleus forskohlii Briq a case study
Ennus Tajuddin Tamboli1, Karishma Chester2, Sayeed Ahmad1
1 Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India
2 Department of Pharmacy, Bansthali Vidyapith, Bansthali University, Tonk, Rajasthan, India
|Date of Submission||13-Apr-2014|
|Date of Decision||03-Jan-2015|
|Date of Acceptance||15-Feb-2015|
|Date of Web Publication||23-Oct-2015|
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard, New Delhi
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: Current trend of commercialization of herbal medicines draw a huge need of maintaining their quality. The declaration of quality, safety and efficacy of medicinal plants as well as poly-herbal formulations has become an important issue. Hence, qualitative and quantitative analysis of herbal drugs and formulations viz., fingerprint profiles and quantification of the various markers become key factors of quality control. Materials and Methods: Present investigation is a detailed report for quality control of well-known herb Coleus forskohlii Briq, which includes physicochemical parameter determination, safety evaluation, microscropical evaluation, and chromatographic fingerprinting as well. Results: Physico-chemical characters were evaluated according to Indian Pharmacopoeia, further microscopic evaluation of transverse section of Coleus reveals that periderm, secondary phloem, and wide secondary xylem cylinder, which occupies major portion of the root fragmentary. Chromatographic fingerprint profiles of Coleus have been generated, and a marker based standardization strategy was adopted; using different analytical technique like high-performance thin layer chromatography, high-performance liquid chromatography and gas chromatography-mass spectroscopy to maintain quality and ensure safety as well as efficacy. Conclusion: These advancements in modern techniques of analysis can lead to effective quality control of Coleus as well as other herbs. Present report can act as pioneer for quality control of modern herbal medicine.
Keywords: Coleus forskohlii, gas chromatography-mass spectroscopy, quality control, standardization, thin layer chromatography
|How to cite this article:|
Tamboli ET, Chester K, Ahmad S. Quality control aspects of herbs and botanicals in developing countries: Coleus forskohlii Briq a case study. J Pharm Bioall Sci 2015;7:254-9
|How to cite this URL:|
Tamboli ET, Chester K, Ahmad S. Quality control aspects of herbs and botanicals in developing countries: Coleus forskohlii Briq a case study. J Pharm Bioall Sci [serial online] 2015 [cited 2020 Nov 26];7:254-9. Available from: https://www.jpbsonline.org/text.asp?2015/7/4/254/168020
The plants are indispensable to man for his life. The three important necessities of life – food, cloth and shelter are supplied to man by plant kingdom and act as a complete storehouse of remedies to cure all kind of disease in man. Men learnt to use plants as healers of different ailments with the beginning of civilisation, since then very vast knowledge concerning therapeutic properties of different plants has accumulated. According to one estimate, 80% of the world population still depends on herbal products for their primary healthcare needs. With the growing interest in plant-based medicine over the last 20 years, herbal medicine has been enjoying people's attention throughout the world.
Plectranthus barabatus (Coleus forskohlii Briq, Family: Lamiaceae) is also an important medicinal plant, which is mainly distributed in few developing countries viz. India, Pakistan, Sri Lanka, and Brazil. Preparations of Coleus species have long been used in Ayurvedic and traditional systems of medicine, particularly in the treatment of epilepsy, conjunctivitis, abdominal colic, etc. The C. forskohlii contains forskolin, which has been demonstrated to posses hypotensive, spasmolytic, cardiotonic and antiplatelet activity.,
Large-scale production of herbal medicine is the current scenario but, along with the commercialization of herbal medicines there is a huge need of maintaining their quality. There are and there should be proper rules and regulations for maintaining the quality standards of herbal medicines. The assertion of quality, safety and efficacy of medicinal plants and poly-herbal formulations has become an important issue. The advancements in modern methods of analysis and the development of their application have made it possible to solve many of these problems. Phytochemical profiles have to be generated and a multiple-marker-based standardization strategy needs to be adopted to minimize batch-to-batch variation and to maintain quality and ensure safety and efficacy.
In present investigation, quality control analysis of C. forskohlii roots was carried out and generated a proper guidelines for the quality control of a medicinal plant, which includes physicochemical parameter determination, safety evaluation, microscropical evaluation, chromatographic fingerprinting using various analytical techniques and quantitative estimation of known marker constituent.
| Materials and Methods|| |
Collection and authentication
The plant materials (fresh/dry roots of C. forskohlii) were collected from Bengaluru (12°58'0"N 77°34'0"E) (India). Further authenticated and voucher specimen (specimen number JH/BNPL/CF2/2010) was deposited in herbarium of Bioactive Natural Product Laboratory for future reference.
Determination of physico-chemical parameters
Foreign organic matter (FOM), loss on drying, ethanol soluble extractive, water soluble extractive, ash, acid-insoluble ash was determined as per the procedure described in Indian Pharmacopoeia 2007.
Preparation of specimens
Samples of different organs were cut and removed from the plant and fixed in formalin (5.0 mL) + acetic acid (5.0 mL) + 70% ethyl alcohol (90 mL). After 24 h of fixing, the specimens were dehydrated with graded series of tertiary butyl alcohol as per the schedule given by Sass, 1940. Infiltration of the specimens was carried out by gradual addition of paraffin wax (melting point 58–60°C) until thiobarbituric acid solution attained super saturation. The specimens were cast in to paraffin blocks.
The paraffin embedded specimens were sectioned with the help of rotary microtome. The thickness of the sections was 10–12 µm. Dewaxing of the sections was by customary procedure. The sections were stained with toluidine blue as per the method published by O'Brien et al. 1964. Glycerine mounted temporary preparations were made for macerated/cleared materials. Powdered materials of different parts were cleared with NaOH and mounted in glycerine medium after staining different cell component were studied and measured.
Microscopic descriptions of tissues are supplemented with micrographs whenever necessary. Photographs of different magnifications were taken with Nikon lab photo 2 microscopic unit. For normal observations bright field was used. For the study of crystals, starch grains and lignified cells, and polarized light was employed. Since these structures have birefringent property, under polarized light they appear bright against dark background. Magnifications of the figures are indicated by the scale-bars. Descriptive terms of the anatomical features are as given in the standard anatomy books.
Evaluation of contaminants
Heavy or toxic metal analysis
The heavy metal analysis for the presence of lead, cadmium, mercury and arsenic was performed for the dried Coleus powder. It was carried out by using atomic absorption spectrometer. Standard linear calibration curve was prepared with absorbance against concentration and concentration of respective metal was calculated in the samples.
The AOAC official method of analysis was followed for the determination of aflatoxins.
Pesticides were analysed in gas chromatography-mass spectroscopy (GC-MS) (Agilent 7890A GC system, USA) by established AOAC official method.
Thin layer chromatographic fingerprinting
The thin layer chromatographic fingerprinting was developed according to the protocol suggested by Tamboli et al.
Gas chromatography-mass spectroscopy fingerprinting
Volatile oil of C. forskohlii was extracted using Clevenger type apparatus. In order to remove any immiscible water droplets, the oil obtained was centrifuged for 15 min at 10,000 rpm and supernatant was collected in fresh vials. For GC-MS analysis, oil was diluted with high-performance liquid chromatography (HPLC) grade hexane and filtered with 0.45 µM polytetrafluoroethylene syringe filter (Axiva Sichem Biotech, Delhi) and further injected to GC-MS.
Gas chromatography-mass spectroscopy analysis
The samples were analyzed on Agilent 7890A GC system coupled with 5975C inert XL EI/CI MSD Model No. G3174A, Agilent Technologies, Wilmington, DE, USA mass spectroscopic system. The system was equipped with a 30 m-long, 0.25 mm i.d. and 0.25 mm film-thickness HP-5MS capillary column. The CTC analysis CombiPAL robotic arm (G6500-CTC-LHS2.PAL system-CH001210757, CTC Analytics AG, Zwingen, Switzerland) was connected to GC/MS for auto sampling system and injector was operated in splitless mode and the injection volume was 500 µL. The inlet temperature was kept at 250°C; the oven temperature was initially kept at 30°C for 2 min. The temperature was then increased to 80°C at a rate of 4°C/min and held at that temperature for 1-min. The temperature was then raised at 6°C/min–250°C and held at the same for 2 min. Helium was as used as carrier gas and flow rate was kept at 1.0 mL/min throughout the analysis. The MS detector was operated in SCAN mode. The headspace was programmed using software employed for operating GC-MS system. The sample vials containing root powder were incubated at 65°C for 900 s the agitator was kept at speed of 500 rpm in order to throughout distribution of heat and avoiding dead spots. Syringe temperature was also kept at 65°C prior to sampling; the samples were injected in split-less mode.
Identification of components
The identification of constituents was performed on the basis of retention time, mass spectral comparison by library (NIST and Wiley) and MS literature data. The relative amount of individual components were calculated based on the GC peak area (MS response) and recorded.
Quantitative estimation of marker compound
Quantitative estimation of forskolin was carried as per the validated HPLC method reported by Tamboli et al. using C18 column as stationary phase and water: Acetonitrile (50:50, v/v) as mobile phase.
| Results and Discussion|| |
Use of medicinal plants has been raised globally in tremendous manner hence, it became necessary to keep check on their quality, efficacy, and safety. In order to determine the quality of raw material concerning medicinal plant products, standardization of medicinal herbs is method of choice. Coleus is also an important medicinal plant being used extensively in treatment of cardiac disorder, glaucoma, and also as anti-obesity as well. Hence, Coleus sample was evaluated as per the physicochemical parameters prescribed by Indian Pharmacopoeia. The experiment was carried out in triplicate and results were summarized [Table 1]. There are several factors on which quality of herbs and herbal product depends. Evaluation of physicochemical parameters for medicinal plant begins with determination of foreign organic material present in raw material. Determination of FOM was prior need. Value of FOM is found in within the limit which is not more than 2%, hence Coleus sample passes the pharmacopoeial limits. Herbal drugs containing moisture are more prone to degradation or deterioration, therefore, determination of moisture among the sample is necessary. The results obtained after determination of loss on drying was 2.07 ± 0.51, which illustrated that samples were properly air dried and contains lesser amount of moisture. Extractive values were determined using solvent alcohol and water, which was 19.48 ± 0.45% for alcohol soluble extractives and 19.75 ± 0.23% for water soluble extractives; however, pharmacopoeial limit for the alcohol soluble and water soluble extractive is not <15% and 18%, respectively. Total ash is the measure of the total amount of material left after burning and includes ash derived from the part of the plant and adherent material. The total ash value for Coleus samples was 5.97 ± 0.21%, which was again in the limit (not more than 15%). The acid-insoluble ash is the residue obtained after boiling the total ash with dilute hydrochloric acid, and burning the remaining insoluble matter. The process measures the amount of silica present, especially in the form of sand and siliceous earth. The value of acid-insoluble ash was 2.10 ± 0.10%, which is lesser than 5% and thus passes the test. The analysis for safety and toxicity evaluation proclaims that the sample of Coleus was free from any biotic and abiotic contaminants viz., aflatoxins, heavy metals and pesticide.
Microscopic features of the root
A thin root measuring 1.1 mm thick, is wavy in transverse section outline. The epidermis and the cortex have been outlined, leaving only small remnants on the surface. A thin layer of periderm comprising three or four layers of cells is seen along the outer part of the root [Figure 1]a. In periderm, cells are compressed into narrow, tangentially much elongated cells. The major portion of the root is occupied by secondary xylem [Figure 1]a. The vessels are more crowded in the central core and the xylem fibres are also thick walled narrow and darkly stained in the centre. A long the outer zone, the vessels become space slightly narrow and diffuse in distribution. The vessels in general are, are angular and thin walled. They are solitary or less frequently in multiples of two. They are 30–80 µm in diameter.
|Figure 1: (a) T. S. of thin root-entire view. (b) T. S. of thick root-one sector. (c) T. S. of thick root-central portion enlarged. (d) T. S. of outer part of the root cortex, secondary phloem and secondary xylem cells (the boundary between the two zones not distinct) (Co: Cortex, CS: Central segments of xylem, FX: Fissured xylem core, FX: Fissured xylem segments, Pe: Periderm, Ph: Phloem, PS: Parenchyma sheath, SE: Slave elements, SPh: Secondary phloem, SX: Secondary xylem, VC: Vessel cluster, Ve: Vessel, X: Xylem elements, XF: Xylem fibres, XP: Xylem parenchyma)|
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Root measuring 3.8 mm radius was studied. The thick root consists of a thin, superficial less conspicuous periderm, narrow but distinct secondary phloem and wide secondary xylem cylinder which occupies major portion of the root [Figure 1]b. The secondary xylem is nearly 3.0 mm in radial width. It can be divided into two distinct zones: Central core of the three fan-shaped dense xylem segments and outer wider zone of the xylem parenchyma and isolated and scattered nests of vessel [Figure 1]c and [Figure 1]d.
The central core of segments are split into three fan-shaped wings lay wide gaps, which are developed due to mechanical stress. The three segments consist of dense by crowded vessels, which are circular, thin walled and wide. They are up to 90 µm in diameter. The sclerenchymatous ground tissue within the segments consists of thick walled lignified cells [Figure 1]c]. The central portion of secondary xylem is evidentlyfirst formed tissue.
The outer wide zone of secondary xylem is produced by the vascular cambium towards the inner side. The derivatives are mostly unlignified, thin walled xylem-parenchymatous cells and small, nests of diffusely scattered vessels. The vessels are angular, thick walled and are up to 100 µm wide. The vessel clusters are ensheathed by a layer of parenchyma cells. Secondary phloem occurs outside xylem cylinder and inner the cortex. It includes mostly parenchyma cells and small groups of sieve-elements, which are thick walled and narrow [Figure 1]c and [Figure 1]d. Starch grains are widely distributed in the xylem parenchyma cells. They are diffuse in distribution and within a cell only 2–4 grains are seen [Figure 2]a. Starch grains are denser in the parenchyma cells that in sheath the vessels [Figure 2]b. The starch grains are simple type spherical and concentric.
|Figure 2: (a) Distribution of starch grains in the xylem parenchyma. (b) Starch grains with parenchyma cell around vessels. (c) Peridem cell in plain view. (d) Starch grains with parenchyma. Fibre and starch grains. (e) Septate fibre. (f) A vessel Long row of vessel members. (g) A vessel element showing end wall perforation and lateral wall pits (SG: Starch grains, Sx: Secondary xylem, Ve: Vessels, X: Xylem, Pe: Periderm, Fi: Fibre, Se: Septum, SFi: Septate fibre, Pi: Pit, PP: Perforation plate, VM: vessel membrane). (h) Vessel element showing end wall perforation and lateral wall pits|
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The powdered preparation of the root exhibits the following components:
Small pieces of periderm tissue are often seen in the powder [Figure 2]c. They are seen in face view (surface view). The cells are rectangular in shape and are thin walled, they occur in regular shape and parallel rows. The cells are 50 µm × 40–70 µm in size.
Cells containing dense accumulation of starch grains are one of the inclusions in the powder [Figure 2]c and [Figure 2]d. The starch grains are simple type; they are spherical in shape with central hilum. The grains are up to 10 µm in diameter.
Long, narrow, and fairly thick walled fibres are very common in the powder. Two types of the fibres were seen in [Figure 2]e and [Figure 1]f.
These fibres have narrow lumen and thick walls. They are nonseptate. They are more than 1.0 mm long and 20 µm thick [Figure 2]e.
These fibres are wider with comparatively thick walls. They have septa dividing the cells into two or more units. The septate fibres are 1.3 mm long and 60 µm wide. The septate fibres have simple pits. They are storage in function [Figure 2]f.
Short cylindrical and wide vessels and vessel elements are frequently seen in the powder [Figure 2]g and [Figure 2]h. They have slightly oblique wide circular simple perforations at the end wall. The lateral walls have horizontally oblong alternate pits [Figure 2]h. The vessel elements are surrounded by parenchyma cells.
The quality of the drug can also be assessed on the basis of the chromatographic fingerprint, in addition high-performance thin layer chromatography (HPTLC), HPLC methods can also provide qualitative and quantitative information about the main active constituents present in the crude drugs. HPTLC is being employed extensively as it enables rapid analysis of herbal extracts with minimum sample clean-up requirement. Chromatographic fingerprinting of methanol extract of Coleus showed good separation of different compounds [Figure 3]. The HPTLC chromatogram and peak data [Table 2] obtained after the scanning of developed plate confirmed separation of forskolin along with 15 other metabolites. Further, quantitative estimation of forskolin using HPLC results showed that the root of Coleus contains 0.285 ± 0.006% w/w of forskolin.
|Figure 3: High performance thin layer chromatography chromatogram of methanol extract of Coleus forskohlii|
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|Table 2: Substances separated in fingerprinting of methanol extract of C. forskohlii|
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The GC-MS anslysis leads to separation of 45 components [Figure 4] and [Table 3]. However, the major constituents present were α-ylangen (1.61%), 1,8-cineole (1.73%), borneol (2.63%), α humulene (3.4%), β pinene (3.61%), camphene (3.68%), n-decanoic acid (3.92%), α-pinene (4.39%), 1-decanol (5.49%), cetene (8.85%), decyl acetate (9.24%), bornyl acetate (16.9%), and decanal (18.4%).
|Figure 4: Gas chromatography-mass spectroscopy chromatogram of volatile oil of Coleus forskohlii|
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|Table 3: Various identified constituents present in volatile oil of C. forskohlii roots|
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Pharmacognostic evaluation of Coleus was also reported by Srivastava et al. 2002 but present report on Coleus is based on advanced guidelines of official books as well as techniques used in present investigation, that is, HPTLC, digital photography for microscopy, GC-MS analysis for volatile constituents are much more reliable. Qualitative and quantitative estimation of methanol extract of Coleus was also carried in present investigation using a validated HPTLC as well as HPLC method however, previous report of quantitative estimation of forskolin by Srivastava et al., 2002 was based on benzene extract. Hence, present investigation provides significant data for quality control of Coleus.
| Conclusion|| |
Present investigation is an emphasised report on quality control and safety evaluation of Coleus roots. The report is enriched with the scientific data obtained after following various standard testing procedures suggested by official books. In addition, the report is also having summarized information about microscopic evaluation of Coleus roots and its powder. The GC-MS analysis was also carried out for the exploration of volatile oil component. Hence, present report is fulfilling the basic needs of quality control of raw materials obtained from plant origin, which can act as pioneer for quality control of modern herbal medicine.
Authors are thankful to All India Council for Technical Education for providing financial assistance and University Grants Commission India for providing meritorious fellowship F.11-26/2008 (BSR) to Mr. Ennus Tajuddin Tamboli for this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]