INTRODUCTION:

Medicinal plants have been used in traditional medicines since ancient times because of their lesser side effects, more accessibility and efficacy. A large number of bioactive compounds are synthesized inside the plants for various purposes. Globally, medicinal plants are being studied in order to develop new molecules for use in pharmacology, neutraceuticals, food supplements, folk medicines etc.

One of such medicinal plant is Terminalia arjuna. It is known worldwide for its excellent cardiotonic effects. Acharya vaghbata introduced it as medicine in cardiovascular disease. In recent time it has been used as expectorant, aphrodisiac, anti dysentric, etc. it has been used as classical medicine for many ailments such as hypertension, bronchitis, asthma, excessive perspiration, and diabetes etc.

Although all parts of T. arjuna have been used as medicine but its bark is used more promniently. Saponins, sterols, minerals, flavonoides, tannins, and polyphenols are major constituents of T. arjuna bark. Many types of amino acids such as histidine, cysteine, and tryosine are also found in T. arjuna. Its stem bark contains triterpenoides such as arjunin, arjunic acid, arjungenin, terminic acid, terminoltin, arjunolic acid; glycosides such as arjunetin, arjunolone, arjunoside, arjunolitin, flavonoides and phenols such as arjunon, luteoline, gallic acid, quercetin, tannin such as punicallin, castalagin, casurin, minerals and trace elements such as Ca, Mg, Al, Zn, Cu and silica [1,2].

The medicinal plants are used at different levels in society in various fields such as in industry, pharmaceuticals, food industry, household etc. So these medicine plants have to be convenient to transport, storage and use along with extended shelf life. So these medicine plants are dried in order to remove the water content and make them easier to use for further purposes.

For drying, different techniques are used such as sun drying, freeze drying, microwave drying, and oven drying, etc. Sun drying is most common method since ancient time and cost effective method. The risk is degradation of quality of samples and direct exposure to environment lead to contamination. Oven drying method that uses thermal energy to remove the moisture of samples. This is one of easiest method for drying and preserving the phytochemicals. Microwave drying uses the electromagnetic radiation that possesses both electic and magnetic fields. The electric field causes simultaneous heating through dipolar rotation; alignment on the electric field of the molecules possesing a permanent or induced dipole moment and ionic induction that cause the oscillation of the molecules and resulting in the fast heating of the samples simultaneously [3]. Freeze drying method is based on the principle of sublimation. Sample is frozen at -80ºC to -20ºC to solidify any liquid in the samples. The frozen sample is subjected to high vaccum which removes the ice by sublimation. Freeze drying yielded higher level of pheolic components. Freeze drying is complex and expensive method but useful for retain delicate and heat sensitive bioactive components of higher value. Different drying techniques cause differentiation in the bioactive compounds in the medicinal plants. So drying method should be chosen carefully in order to retain the maximum amount of bioactive compounds along with other desired benefits. But there are a few studies available about the impact of various drying techniques on bioactive compounds of samples. So the purpose of this research is to find out the effects of different drying techniques such as microwave drying, oven drying, freeze drying, etc on the bioactive compounds of T. arjuna stem bark.

Sample Collection:

Arjuna stem bark were procured from the Medical Aromatic and Under Utilized Plant Section Dept. of Plant Breeding, CCS HAU, Hisar.

Sample preparation:

Sorting:

Fresh, black bark were collected and seperable parts were discarded before washing and washed with 3 times with distilled water to remove unwanted dirt particles.

Drying techniques:

The washed stem bark of Arjuna was subjected to four different methods.

Sun drying:

Arjuna stem bark were distributed on the stainless steel trays and dried under direct sunlight at temperature between 25°C and 30°C, for 5 days with about 36 hrs of daylight.

Oven drying:

Arjuna stem bark were distributed on the stainless steel trays and dried in oven at temperature 50ºC, for 2 days.

Freeze drying:

In freezing, the Arjuna stem bark were put in the lyophilized condition at –20°C freezer until weight become constant.

Microwave drying:

Arjuna stem bark were placed in a single layer in a Pyrex petri dish and heated in microwave oven for 20 minutes until weight become constant. After heating, the Arjuna bark were allowed to cool in desiccator.

Sample extraction:

Preparation of plant extracts:

All the types of dried powdered samples (15g each) of Terminalia arjuna were subjected to soxhlet extraction using methanol (100 ml each). All the extracts were filtered through Whatman No.1 filter paper and then concentrated under reduced pressure in a rotary evaporator. The dried extracts were then weighed. Extraction yield was calculated by subtracting the dried weight of plant material residues after extraction from the weight of the original plant material. The methanolic extracts thus obtained were stored at refrigeration temperature in sterile vials for further experiments [4,5].

Qualitative analysis:

Phytochemical analysis of the test sample was carried out according to standard methods [6-9].

Salkowski reaction test for phytosterols:

0.5 mL of T.arjuna stem bark extracts solution were added followed by addition 1mL concentrated H₂SO₄from the sides of the test tube. The presence of reddish brown colour in test tube indicates the presence of phytosterols.

Liebermann-Burchard’s test for triterpenoids:

T.arjuna stem bark extracts were treated with few drops of acetic anhydride. The prepared samples were boiled and then cooled in room temperture. Further few drop of concentrated H₂SO₄ was added from the sides of the test tube. A redish brown coloration at the surface showed the presence of triterpenoids.

Foam test for saponins:

Small amount of T.arjuna stem bark extracts were mixed with distilled water and shaken vigorously for stable persistant froth. The formation of foam indicated presence of saponins in the test tube.

Dragendroff’s test for alkaloids:

T.arjuna stem bark extracts were dissolved in chloroform. Chloroform was evaporated and the residue was acidified by adding few drops of Dragendroff’s reagent (Potassium bismuth iodide). Formation of appearance of orange red precipitate indicated the presence of alkaloids.

Molisch’s test for carbohydrates:

T.arjuna stem bark extracts were mixed with concentrated H₂SO₄to form layers. Formation of reddish violet ring showed the presence of carbohydrates.

Quantitative Analysis:

Determination of total alkaloids:

Total alkaloid content estimated by Ajiboye et al. [10]. 5g of the sample was extracted in 200ml of 10% acetic acid in methanol. The solution was filtered through muslin cloth and the extract was concentrated on a water bath to 1:3 ratio of the original volume. After that concentrated ammonium hydroxide was added drop wise to the solution until the precipitation was completed. The whole solution was allowed settle and the precipitated was collected and washed with dilute ammonium hydroxide and filtered. The residue is the alkaloid, which was dried and weighed [10,11].

Determination of proteins:

Protein content of T.arjuna stem bark extracts were determined by Micro-Kjeldhal method described by AOAC [12]. Weigh 1g of T.arjuna stem bark powders by digesting in 20 ml concentrated sulphuric acid containing a pinch of catalyst mixture for 2-3h at 100ºC. Digested contents were cooled, diluted and volume made upto 100 ml with distilled water. 10 ml aliquot of digested was taken in beaker and 20 ml alkali was added and steam distilled. The liberated ammonia was distilled into 10 ml boric acid solution containing a few drops of mixed indicator. The distillate was titrated with N /100 hydrochloric acid to determine the ammonia absorbed in boric acid.

The % nitrogen was calculted by formula and % protein was estimated by multipiying with factor 6.25.

S – B x v x 100 x 0.00014

Nitrogen (%) = ^{--------------------------------------------------------------------------}

V1 W

Total Protein = % Nitrogen ×6.25

Where, W is weight of sample (g), V is the volume of extract made (ml), V₁is volume of aliquot taken for distillation, S is volume of standard acid 0.01 N H₂SO₄ used for titration (ml), B is volume of 0.01 N HCL used for blank (ml).

Determination of total flavonoid content:

Aluminum chloride colorimetric method was used for flavonoids determination by Wang and Jiao [13]. About 1 mL of the T.arjuna stem bark extracts of different concentration were mixed with 3 mL of methanol, 0.2 mL of aluminum chloride, 0.2 mL of 1 mol/L potassium acetate and 5.6 mL of distilled water. It remained at room temperature for 30 min. The absorbance of the reaction mixture was measured at 415 nm with spectrophotometer against methanol served as blank. The total flavonoid content in T.arjuna stem bark in methanol was calculated by the following equation:

c × v

C = ^{---------------}

Where, C is total flavonoid content, mg/g plant extract, in quercetin equivalent; c is the concentration of quercetin established from the calibration curve in mg/mL, V is the volume of extract in mL, and m is the weight of crude plant extract in g.

Determination of total phenol content:

The total phenols were determined as per the method given by Hagerman et al. [14] using Folin-Ciocalteu reagent. Weigh 100mg of T.arjuna stem bark extract and dissolved in 100 ml of double distilled water (DDW). 1 ml of this solution was transferred to a test tube, and add 0.5 ml 2N of the Folin-Ciocalteu reagent and 1.5 ml 20% of Na₂CO₃ solutions were added and made up the volume up to 8 ml and allowed to stand for 2 hours and measured at 765 nm. A standard calibration curve of galic acid using different concentrations was prepared by measuring the optical density at 765nm. The concentration of phenols in samples were expressed as mg/100g [10,14]

Antioxidant activity determination by DPPH free radical scavenging assay:

The radical scavenging activity of the extract was measured by the method described by Barros et al. [15]. DPPH reacts with antioxidants compounds which can donate hydrogen and reduce DPPH. The change in color from violet to light yellow was measured optical density at 517nm using visible light spectroscopy. 1 mL of T.arjuna stem bark extracts were placed into test tubes and 0.5 mL of 1 mM/L DPPH solution in methanol was added. The test tubes were incubated for 15 minutes at room temperature and the optical density was measured at 517nm against methanol. The DPPH radical scavenging activity percentage was calculated by using the following formula:

DPPH radical scavenging Activity (%)

= A_control - A_extract / A_control×100.

Antimicrobial activity using agar well diffusion method:

The antimicrobial activity of methanolic extract T. arjuna was evaluated by agar well diffusion method Perez et al. [16]. The bacterial culture of 0.5 McFarland Standard (~2 × 108 colony forming units (CFU)/ml) was uniformly spread on the surface of the nutrient agar plates using sterile cotton swabs [17]. The wells were punched with the cork borer (6 mm) in the agar. Approximately 50 mL of the crude extract (50 mg/ml) of bark were added into the wells, allowed to stand at room temperature for about 2 h and incubated at 37 ºC. After 24 h of incubation, the zone of inhibition was measured using a Hi Antibiotic Zone scale-C (Himedia Biosciences, Mumbai (India)). Erythromycin (10 mg) was used as a positive control in case of bacterial strains. Methanol (solvent) was used as solvent control. The tests were performed in duplicates and results were recorded as Mean ± SD.

RESULT AND DISCUSSION:

Preliminary phytochemical analysis of Terminalia arjuna extract dried by different methods are given in table 1.

The Preliminary phytochemical analysis conducted on differently dried extract of Terminalia arjuna stem bark revealed the presence of phytochemicals like alkaloids, saponins, triterpenoids and phytosterols which are known to exhibit medicinal as well as physiological activities. It is also reported the presence of phytochemicals, such as alkaloids, phenols, saponins, triterpenoids and phytosterols in T. arjuna stem bark [6].

Quantitative Analysis:

Total phenolic content:

The results of the total phenolic contents of different dried T. arjuna stem bark extracts are presented in table 2 and (figure 1). The phenolic content of freeze, microwave, oven and sun dried samples were as follows: 337.6, 302.6, 256.8 and 204.4 mg/g DW respectively. The phenolic content in the oven-dried and sun-dried samples were significantly lower than as comparison to microwave dried and frezze dried samples. The results of this study revealed that freeze drying technique was most efficient technique and sun drying technique was most detrimental technique among all the drying techniques. Similar results were found in which state that dehydration at low temperatures leads to retention of higher number of bioactive compounds in plants [18, 19]. It also revealed that thermal processing in oven and sun drying techniques ruptures the cell structure of T. arjuna bark which may lead to migration of bioactive components and their loss [20]. Furthermore higher phenolic content was found in microwave drying as compare to sun and oven drying in Carissa spinarum fruit. This may be due to less heating duration in microwave drying [21,22]. In addition various researchers have reported that the breakdown of bioactive compounds during thermal processing is higher. This degradation could be due to the activity of polyphenol oxidases which have ability to degrade phenolic compounds [23,19].

Fig. 1 Total Phenol content of different dried T. arjuna extracts

Total flavonoid Content:

The value of TFC in bark extract was found to be freeze-dried (278.4 mg) > microwave dried (265.86 mg) > Oven dried (255.36 mg) > Sun dried (226.1 mg) in table 2 and (figure 2). TFC of freeze dried T. arjuna bark were found to be more in the present study, since heating causes breakdown of some bioactive components which affect cell wall intergrity and leads to migration of some flavonoids component. There are some reports available which showed that there was less degradation of flavonoid content in microwave drying as compared to sun and oven drying. This is might be due to high output power of microwave and less exposure to atmospheric oxygen while in freeze drying the flavonoid content increases because of increase in extractability and hydrolysis of quercetin [21,24].

Alkaloid content:

Alkaloid content in freeze dried T. arjuna bark had 0.96%, which was higher than 0.94% (microwave), 0.93% (oven) and 0.91% for sun dried samples in table 2(figure 3). The highest percentage of alkaloid content was found in the freeze dried T. arjuna bark whereas the lowest alkaloid content was found in sun dried of T. arjuna bark. This is because alkaloids are thermolabile and not stable at higher temperature. It is also state that they undergo degradation or decomposition on exposure to air, light, moisture and heat or chemicals [25]. Another study which also confirmed that freeze drying has the potential of retaining alkaloid content as compared to other drying techniques in leaves of Carissa edulis [26].

Fig. 2 Total Flavonoid content of different dried T. arjuna extracts

Fig. 3 Total Alkaloid content of different dried T. arjuna extracts

Protein content:

Protein content was found to be 3.10%, 2.57%, 2.52%, 2.02% for freeze, microwave, oven and sun dried samples respectively mentioned in table 2 (figure 4). It is also reported that drying could cause changes in chemical structure of polyphenols or cause them to adhere together with other plant components (proteins) leads to extraction. Thermal process results in rupture of cell structures and deterioration of bio-active components which leads to denaturation of proteins [27].

Fig. 4 Total Protein content of different dried T. arjuna extracts

Antioxidant activity:

Highest DPPH scavenging activity was observed in freeze dried T. arjuna bark (86.53 %), followed by microwave dried (83.64%), oven dried (74.19%), sun dried (69.22%) in T. arjuna bark samples, respectively (Table 2) (figure 5). It is well correlated with the results reported by Mudau and Ngezimana [28] that freeze drying has the highest potential of retaining phenolic compounds and antioxidant activities of most samples as compared to other drying methods. It is also reported that freeze dried leaves recorded the highest antioxidant activity among the dried dandelion leaves [19].

Fig.5

Antimicrobial activity:

The results from the agar well diffusion method and indicated that extract of freeze dried bark exhibited more antimicrobial activity as compared to methanolic extract of oven, microwave and sun dried bark with diameter of zone of inhibition were 16.9±0.6 mm, 16.5±0.7 mm, 16.0 ± 0.8 mm, 16.4±0.7 mm, 16.0±0.6 mm, 16.0±0.6 mm in bark (Table 3) against B. subtilis, S. aureus, E. coli, K. pneumoniae, P. aeruginosa, S. typhi respectively (figure 6). The methanolic extract of all dried sample exhibited good antimicrobial activity against all the tested bacterial strains. The antibacterial activity of T. arjuna were in agreement with study in which methanolic extract of T. arjuna bark showed comparative antibacterial activity against Staphylococus aereus, Bacillus megaterium, Bacillus subtilis, Salmonella typhi, Escherichia coli and Pseudomonas aeruginosa [29].

CONFLICT OF INTEREST:

We have no conflict of interest.

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Received on 12.10.2018 Modified on 17.11.2018

Research J. Pharm. and Tech. 2019; 12(5):2372-2378.

DOI: 10.5958/0974-360X.2019.00397.4

S. No	Name of compound	Name of the test	Oven dried	Sun Dried	Microwave dried	Freeze Dried
1	Alkaloid	Dragendroffs test	+ ve	+ ve	+ ve	+ ve
2	Saponin	Foam test	+ ve	+ ve	+ ve	+ ve
3	Triterpenoid	Liebermanne Burchard's test	+ ve	+ ve	+ ve	+ ve
4	Phytosterol	Salkowski test	+ ve	+ ve	+ ve	+ ve
5	Carbohydrate	Benedicts	- ve	- ve	-ve	- ve

Methods	Total Phenol (mg/g)	Total Flavonoid (mg/g)	DPPH (%)	Alkaloid (%)	Protein (%)
Sun Dried	204±1.44	226.1±1.05	69.22±0.28	0.91±0.006	2.02±0.015
Oven Dried	256.86±1.0	255.36±1.07	74.19±0.07	0.93±.003	2.52±0.042
Microwave Dried	302.6±1.30	265.86±1.03	83.64±0.20	0.94±.010	2.87±0.065
Freeze Dried	337.6±1.7	278.4 ±1.05	86.53±0.50	0.96±.009	3.10±0.015

Extract	Diameter of zone of Inhibition (mm)
Extract	*B.subtilus*	*P.aeruginosa*	*S. aureus*	*E. coli*	*S. typhi*	*K. penumoniae*
Sun Dried	10.74±0.41	12.38±0.42	13.65±0.47	12.83±0.42	11.38±0.47	11.70±0.44
Oven Dried	14.50±0.26	13.83±0.15	14.53±0.38	13.93±0.71	13.50±0.40	13.20±0.44
Microwave Dried	14.57±0.51	14.47±0.50	15.40±0.44	14.43±0.60	12.27±0.55	13.83±0.61
Freeze Dried	17.13±0.32	16.70±0.3	17.20 ±0.75	16.40±0.1	16.00±0.1	16.67±0.61