authentication of h. enneaspermus (l.) f....

Chapter 4

Authentication of H. enneaspermus (L.) F. Muell

Chapter 4 Authentication of H. enneaspermus

In vitro culture and bioactivity studies on H. enneaspermus 54

4.1. Introduction

Standardization of raw drugs is essential in order to assess the quality of herbal

products. According to WHO guidelines, herbal products need to be standardized with

respect to safety before releasing it into the market. Herbal medicinal products may vary in

composition and properties and reports on the decrease in quality and efficacy of the herbal

products as experienced earlier has attracted the attention of many regulatory agencies for the

standardization of herbal formulations. Moreover, many dangerous and lethal side effects

have been reported, including direct toxic effects, allergic reactions, effects from

contaminants, etc (Vaidya and Devasagayam, 2007). In this milieu, proper identification and

quality assurance is an essential pre-requisite to ensure reproducible quality of herbal

medicine, which contributes to its safety and efficacy (Zafar, 2005).

Hybanthus enneaspermus (L.) F. Muell. (Syn. Ionidium suffruticosum Ging.) of

Violaceae is popularly known as ‘Spade flower’, ‘pink ladies slipper’ (English), ‘ratanpurus’

(Hindi), ‘padmacarini’ (Sanskrit), ‘orilathamara’ (Malayalam), ‘orithaltamarai’ (Tamil) and

‘purusharathnam’ (Telugu). In Ayurveda, the botanical identity of ‘padmacarini’ is

controversial. Some physicians have considered it as Nervilia aragoana (Bhogaonkar and

Devarkar, 2007) or Habenaria grandiflora (Moss, 1980). However, Kirtikar and Basu

(1918), Nadkarni (1954) and Chopra et al. (1956) have described ‘padmacarini’ as H.

enneaspermus. The plant is widely used in traditional systems of medicine such as Ayurveda

and Siddha. The ethnomedicinal uses of this plant have increased significantly. It has

aphrodisiac, demulcent, tonic and diuretic actions and is used against urinary infections,



diarrhea, leucorrhea, dysuria inflammation and sterility (Tripathy et al., 2009). The plant is

under threat in the natural habitat due to over exploitation by natural healers, overgrazing by

animals, seasonal habitat, sporadic distribution and poor germination frequency of seeds

(Prakash et al., 1999; Sonappanavar and Jayaraj, 2011). The non-availability and controversy

in botanical identity may lead to adulteration in the genuine drug. Moreover, it is difficult to

identify H. enneaspermus from its co-existing weeds in the absence of flowers. Therefore, a

detailed authentication of the drug is quite essential. Retnam and De Britto (2007a) have

studied some aspects of H. enneaspermus’s pharmacognostic characterization whereas,

Bhogaonkar and Devarkar (2007) described the pharmacognostic features of Habenaria

grandiflora and Nervilia aragona, the other two species considered as ‘padmacarini’ in

Ayurveda. The present chapter describes a more efficient standardization protocol including

chemical and biological assays for the authentication of drug.

4.2. Materials and Methods

4.2.1. Collection and identification of H. enneaspermus

Hybanthus enneaspermus was collected from Thanjavoor, Tamil Nadu during the

month of February when the plants were in active growth and bloomed well. Whole plants

were uprooted and the soil and other particles adhered on the plants were removed.

Identification of the plants was confirmed with the help of authentic literature (Gamble, 1935;

Sivarajan and Balachandran, 1994).



4.2.2. Evaluation of Macromorphology

Morphological features of the plant parts were noted as per the method described for

taxonomic evaluation of plants. Features of root, leaf, stem, flowers and fruits were analyzed.

4.2.3. Microscopic Evaluation

Hand sections of the stem, root and leaves were taken and observed under the

microscope. Anatomical peculiarities of different parts of the species and the various

elements present in the dried powder samples were studied.

4.2.4. Physico-chemical Analysis

4.2.4.1. Determination of moisture content

Moisture content (percentage loss on drying) was determined gravimetrically in

which 100 g whole plant sample was dried in an oven at 100-105οC until 2 consecutive

weights did not differ by more than 5 mg.

4.2.4.2. Determination of foreign matter

Dried samples of H. enneaspermus (100 g) were weighed out and spread into a thin

layer on a white paper. Foreign materials like stones, extraneous parts or other contaminants

were separated by using a hand lens and forceps. Weight of the foreign materials was taken

and its percentage with respect to the sample was calculated.

% Foreign material = Weight of the foreign material (g) / Weight of the sample taken (g) x 100.



4.2.4.3. Solvent extractive values

The extractive values were noted in alcohol and water. Coarsely powered air dried

whole plant samples of H. enneaspermus (4 g) were taken out and macerated with 100 ml

solvent for 6 hours. The mixture was filtered and 25 ml of the filtrate was taken in a flat

bottomed dish and evaporated to dryness at 105οC for 6 hours. Weight of the extract was

taken immediately after cooling.

4.2.4.4. Ash content

Air dried and powdered sample of H. enneaspermus (3g) was taken in a sintered

crucible and incinerated in a Bunsen burner at 500οC, until a white ash was obtained. After

cooling, the weight of the sample was taken and the procedure was repeated until the weight

became constant (Indian Pharmacopoeia, 1985).

% Total Ash = Weight of air dried drug (g) / Weight of ash (g) x 100.

4.2.4.5. Determination of acid insoluble ash:

Ash obtained from a known amount of air dried plant sample was boiled with 25 ml

dilute HCl for 5 minutes. The solution was filtered through an ashless filter paper and the

residue incinerated along with ashless filter paper at 500-600°C in a Bunsen burner. The

percentage of acid insoluble ash with reference to air dried drug was calculated (Indian

Pharmacopoeia, 1996).



4.2.4.6. Determination of water soluble ash:

The ash obtained from the air dried plant sample was boiled for 5 min. with 25 ml of

water. The insoluble matter was collected on ashless filter paper and washed with hot water.

It was then transferred into a silica crucible and ignited. The weight of insoluble matter was

subtracted from the weight of the total ash and the difference in weight was taken as water

soluble ash.

4.2.4.7. pH values

One and ten percentage aqueous solutions (10 ml) of the dry powder were prepared and the

pH was measured with a digital pH meter.

4.2.5. Chemical evaluation

Whole plant samples of H. enneaspermus were washed and dried in an oven at 45°C.

The dried samples were powdered and successively extracted as per the procedure mentioned

in the Chapter 3 (3.6.1). Preliminary screening of the phytochemical constituents present in

the extracts was conducted by class identification tests. The presence or absence of different

phytoconstituents viz., triterpenoids, alkaloids, steroids, sugars, tannins, glycosides and

flavonoids etc. were detected by the usual standardized methods (Harbone, 1973). Detailed

methodology for phytochemical screening is described in the Chapter 3 (3.6.2). During class

identification tests, methanol extract showed the presence of the maximum number of

compounds compared to chloroform and petroleum ether extracts. Hence, High Performance

Thin Layer chromatography (HPTLC) profiling of the methanol extract was conducted using



CAMAG HPTLC system installed in Tropical Botanic Garden and Research Institute,

Palode, Thiruvananthapuram, Kerala as described in Chapter 3 (3.6.3).

4.2.6. Biological evaluation

Biological parameters such as total reducing power (antioxidant activity) and

cytotoxicity of the methanol extract of the plant were analyzed using in vitro methods. For

the determination of reducing power, various concentrations of the extract (5-500 µg/ml) in

methanol (1 ml) were mixed with equal volumes of (2.5 ml) phosphate buffer (0.2M, pH -

6.6) and potassium ferricyanide (1%). The mixture was incubated at 50oC for 20 min and

then 10% trichloro acetic acid (2.5 ml) was added and centrifuged at 1000 rpm for 10 min.

Upper layer of the solution (2.5 ml) was mixed with deionized water (2.5 ml) and 0.1% ferric

chloride (0.5 ml). Absorbance of the mixture was read at 700 nm in a UV-VIS

spectrophotometer (Shimadzu UV-1700). Ascorbic acid was used as the positive control

(Oyaizu, 1986).

Cytotoxic potential of the methanol extract was evaluated in vitro using trypan blue

dye exclusion method with Daltons Lymphoma Ascites (DLA) cell lines. Viable suspension

of DLA cells (1x106 cells in 0.1 ml) was added to vials containing various concentration of

the extract (0, 10, 20, 50, 100, 200, 250 and 300 µg/ml) and made upto 1ml using PBS. The

vial which received only cell suspension was taken as the control. The vials were incubated

for 3 hours at 37OC and then treated 0.1ml of 1% trypan blue and kept for 2-3 minutes. The

number of stained and unstained cells was counted separately by using haemocytometer.



Dead cells were taken up the blue colour of trypan blue while live cells do not take up the

dye. Percentage of cell death was calculated to evaluate the cytotoxic effect (Talwar 1983).

4.3. Results and Discussion

Herbal raw materials are prone to a lot of variation due to several factors, such as

identity of the plants, seasonal, ecotypic, genotypic and chemotypic variations, drying and

storage conditions and the presence of xenobiotics (Nikam et al., 2012). Standardization of

herbal raw drugs include passport data of raw plant drugs, botanical authentication which

include macroscopic and microscopic examinations, physical parameters like moisture

content, ash value, extractive value etc, identification of chemical composition and

determination of biological activity of the whole plant (Kulkarni et al., 2014). The data

compiled during the current study will be a valuable tool for the authentication of H.

enneaspermus.

4.3.1. Macromorphological features

Macromorphological (organoleptic) characters are of primary importance because it

gives information on identity, purity and quality of crude drugs. It involves identification of

the plant, plant part and the raw drug by studying their external characters such as habit, size,

shape, colour, foliar and floral characters (Venkat et al., 2010). Hybanthus enneaspermus is a

sub-erect or erect herb with somewhat woody base and spreading branches. Root system is

well developed with numerous thin and hairy lateral roots that arise from the main primary

root (0.3-0.7cm diameter). Leaves are simple, alternate, subsessile, linear to lanceolate, 10-20

mm long and 3-7 mm wide. Flowers are solitary, pink coloured and the lower petal is large



(4-7 mm), broadly spatulate, clawed and prominently 3 veined (Plate 4.1. A). Stamens are

five, dimorphic and the anterior filaments appendaged. Capsules are subglobose, 4-5mm in

diameter, loculicidally and elastically 3 valvate.

4.3.2. Microscopic features

Microscopic evaluation is essential in the initial identification of herbs, as well as, in

identifying small fragments of crude drugs. It is needed to determine the correct species

and/or that the correct part of the species is present. Moreover, microscopic characters are

very important, especially when different parts of the same plant are to be used for different

treatments (Kunle et al., 2012). Anatomical features of stem, root and leaf of H.

enneaspermus were studied by observing the cross sections (Plate 4.1. B- D). Distinguishing

microscopic observations of the stem are: the vascular cylinder consists of 1 or 2 layers of

discontinuous patches of perivascular sclereids, 4-7 layered phloem and closed dense

cylinder of xylem. Pith was broad and parenchymatous. Cross section of the root showed a

narrow zone of phellem followed by a parenchymatous cortex, small radial masses of

secondary phloem and a dense zone of secondary xylem. Leaf anatomy consists of prominent

midrib seen on both sides with collateral vascular bundles. Stomata are anisocytic and

prominent on abaxial side with a frequency of 112/mm2 (Plate 4.1. E- F). Powder analysis

plays an important part in microscopic analysis of a drug. Dried whole plant powder of H.

enneaspermus showed vessel elements (spiral, scalariform, and annular), sclereids, tracheids

and stomata.



4.3.3. Physico-chemical parameters

Various physico-chemical parameters of H. enneaspermus were analyzed (Table 4.1.)

4.3.3.1. Moisture content

Determination of moisture content helps to reduce errors during the estimation of the

actual weight of drug material. Drying plays an important role in determining quality as well

as purity of the material. Low moisture content in a plant material suggests better stability

against degradation of product. H. enneaspermus showed 67.5% w/w moisture content and

hence the solid content became 32.5% w/w.

4.3.3.2. Foreign matter

During collection of raw drugs some amounts of related parts of plant or other plants, soil etc

may come along with the drugs. Dried samples (100g) of H. enneaspermus showed 2.5 g

foreign matter.

4.3.3.3. Extractive values

Dried samples of H. enneaspermus showed 12% w/w water extractive and 8% w/w

methanol extractive, which are indicative weights of the extractable phytochemicals present

in the crude drug under different solvent environment.



4.3.3.4. Ash values

Incineration of plant parts generates ash which comprises inorganic matter. When

treated with HCl, it results in acid-insoluble ash which consists mainly of silica and may be

used as a measure of soil present (Kunle et al., 2012). Samples of H. enneaspermus showed

the following ash values: total ash - 14.8 %w/w, acid insoluble ash - 50 % w/w, water soluble

ash - 19.1% w/w.

4.3.3.5. pH values

It helps to know the acidic/basic nature of the sample. 1% aqueous solution of H.

enneaspermus showed the pH value of 6.33 and 10% aqueous solution showed 6.30.

Table 4.1. Physico-chemical parameters of H. enneaspermus.

Sl. No. Parameter Values (%w/w) ± S. E.

1 Moisture content 67.5 ± 0.23

2 Solid content 32.5 ± 0 .56

3 Total ash 14.8 ± 0.92

4 Acid insoluble ash 50 ± 0.48

5 Water soluble ash 19.1 ± 0.65

6 Water extractive 12 ± 1.03

7 Methanol extractive 8 ± 1.08

Data represents the means (± S. E) of 5 samples each.



4.3.4. Chemical evaluation

Chemical and chromatographic techniques are widely used in the identification of herbal

material or extract, because its therapeutic potential depends on the phytochemical

constituents present in the material. Chromatographic fingerprints provide information on the

presence of adulterants, pesticide content, mycotoxins etc. Qualitative phytochemical

analysis of H. enneaspermus was conducted with extracts obtained from successive soxhlet

extraction of the dried and powdered plant samples (Table 4. 2). Petroleum ether, chloroform

and methanol extracts were tested for the presence of compounds such as flavanoids,

coumarins, tannins, phenols, steroids, alkaloids, anthraquinones, quinones, arbohydrates and

proteins. Methanol extract revealed the presence of all the compounds. Whereas, chloroform

extract showed the absence of steroids, carbohydrates and proteins. Petroleum ether extract

showed the presence of coumarins and phenols only. The colour reaction tests help to

determine the identity of the drug and doable adulteration.

High Performance Thin Layer Chromatography (HPTLC) is a valuable quality

assessment tool for the evaluation of herbal drugs. The technique is widely used in the

pharmaceutical industry for quality control of herbs and health products, identification and

detection of adulterants or substitutes in herbal products which also helps in the identification

of pesticide contents and Mycotoxins (Nikam et al., 2012). HPTLC profiling of the methanol

extract detected the presence of 10 distinct bands (Rf: 0.03, 0.12, 0.20, 0. 25, 0.37, 0.29, 0.53,

0.69, 0.78 and 0.84). However, compounds with Rf values 0.03 and 0.53 showed more

intense bands (Plate 4. 2).



Table 4.2. Phytochemical profile of successive extracts of H. enneaspermus.

Sl. No. Extract

Test

Petroleum

ether

Chloroform Methanol

1 Flavanoids - + +

2 Coumarins + + +

3 Tannins - + +

4 Phenols + + +

5 Steroids - - +

6 Alkaloids - + +

7 Anthraquinones - + +

8 Quinones - + +

9 Carbohydrates - - +

10 Proteins - - +

‘+’ indicate presence and ‘-’ indicate absence of compounds



4.3.5. Biological evaluation

Activity based standardization of herbal drugs has been successfully followed for the

authentication of genuine drug. The underlying concept in such assays is mainly to correlate

either pharmacological activity of the herbal drug with specific chemical markers or

inhibitory activity of the herbal drug with a particular disease related enzyme. In the latter

case, the concentration of herbal drug required to inhibit 50% of a fixed amount of enzyme

activity (IC50 value) and/or effective concentration of the herbal drug at which half maximal

value of enzyme activity (EC50) occurs, was exploited. This is because, under defined

conditions, an herbal drug gives reproducible IC50 or EC50values, and can be reliably used for

activity-based standardization (Sumantran, 2010). In the present study in H. enneaspermus,

biological activities such as antioxidant (reducing power) and cytotoxic potential was

analysed using in vitro methods. During the evaluation of reducing power of the methanol

extract, the absorbance (at 700 nm) of samples was increased with increase in concentration,

which indicated the reducing power (antioxidant potential) of the extract (Table 4. A3.). The

concentration of the extract which gave 0.5 absorbance at 700 nm, i.e., IC50 value of

methanol extract was 397 µg/ml. The methanol extract of H. enneaspermus also showed

significant cytotoxic activity under in vitro conditions (Table 4. 4.). The concentration

required for 50% cell death (IC50) was found to be 223 µg/ml. The IC50 values evolved during

the bioactivity studies are reproducible and the assay can be used in the activity-based

standardization of H. enneaspermus.



Table 4. 3. Reducing power activity of the methanol extract of H. enneaspermus

Data represents the means (± S. E) of 3 replications.

Table 4. 4. Cytotoxicity of the methanol extract of H. enneaspermus towards DLA cells.

Sl. No. Concentration (µg/ml) % cytotoxicity ± SE

1 10 5 ± 0.40

2 25 18 ± 0.43

3 50 26 ± 0.39

4 100 30 ± 0.37

5 200 44 ± 0.71

6 250 56 ± 0.42

7 300 59 ± 0.51

Values are mean ± S. E. of triplicate analysis

The chances of adulteration – both intentional and unintentional – are very high in H.

enneaspermus, mainly because of its rarity, controversy in botanical identity and its

morphological similarity with other co-existing weeds. The pharmacognostical data

developed in the present study using macroscopic, microscopic, physico-chemical,

phytochemical and biological parameters can be utilized to identify the genuine drug, H.

enneaspermus.

Sl. No. Concentration (µg/ml) Absorbance at 700 nm ± SE

1 5 0.07 ± 1.82

2 10 0.12 ± 1.31

3 50 0.17 ± 0.89

4 100 0.24 ± 1.65

5 250 0.38 ± 1.84

6 500 0.63 ± 1.25

authentication of h. enneaspermus (l.) f....

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