Proximate Composition and Antioxidant Potential of Fruits of Zanthoxylum Alatum Roxb. (Tooth Ache Tree) and its Acceptability Assessment in Food Products

Proximate composition of whole dried fruits of Zanthoxylum alatum, its seed and seed coat were analyzed and essential oil of seed coat was extracted and evaluated for total phenolic and antioxidant content. To assess acceptability of seed coat powder as a flavouring agent, different concentration of roasted seed coat powder were incorporated in selected Indian foods. Organoleptic evaluation of products was done by panel members. Results revealed that crude protein of whole dried fruits, seed and seed coat were 2.9%, 2.1% and 2.6% respectively. Essential oil extracted from seed coat exhibited good antioxidant activity. Food products prepared with roasted seed coat powder were highly acceptable as compare to control and scores were significantly higher at (p>0.01) paired ttest.


INTRODUCTION
The genus Zanthoxylum from family Rutaceae has 200 species, among them Zanthoxylum alatum Roxb.locally known as 'Timur" which is an armed, erect shrub or a small tree (6 m tall or more), with dense foliage, found in the valleys of sub-tropical Himalayas.It also grows in the Eastern Ghats in Orissa and Andhra Pradesh at 1200m and the lesser Himalayan range in the northeastern part of India (e.g., Naga Hills, Meghalaya, Mizoram, and Manipur).
Fruits of Zanthoxylum and their pericarps are used as a peppery spice in both sweet and savory preparations.It is one of the traditional ingredients in the Chinese spice mixture five-spice powder along with star anise, fennel, clove, and cinammon.It has a unique aroma and flavour that is not hot or pungent like black, white or chili peppers.Instead, it has slight lemony overtones and creates a tingly numbness in the mouth.In India, localities of Kuamaon region use Timur fruit in the form of condiments, spices and medicine.
In the preparation of certain traditional dishes, the use of Timur as a flavouring agent or spice is very popular.Ground, roasted Timur is used to make infused oil and is also paired with salt to make a flavorful seasoning.In view of several medicine properties of Timur and its unique taste, flavor and mouth feel the present study reports proximate composition and antioxidant of seeds of Zanthoxylum alatum along with its usage in various food products

Procurement of plant material
Dried samples of Timur were procured from Krishi Vigyan Kendra of Pittoragarh district, Uttarakhand.Samples were cleaned well and seed and seed coat of whole Timur were separated.Whole Timur fruit (T), seed (S) and seed coat (SC) were weighed and transferred to poly bags and sealed in polythene pouches.

Proximate Analysis
All the three samples i.e.T, S and SC were analysed in triplicate for proximate composition.Samples of T, S and SC were ground using electronic food grinder before analysis and transferred to plastic containers.Moisture content was estimated by oven dry method.Protein content was measured by Kjeldahl procedure of total nitrogen using standard methods.Fat content was extracted from samples with petroleum ether (40-60) by Soxhlet apparatus, while ash content was measured by charring the sample in hot plate and placing in muffle furnace at 600 o C for at least 3 hours then cooled in desiccators and weighed.The ash was almost white or greyish white in colour.Crude fibre was determined by treating oil-free sample by sulphuric acid (0.26 N) and potassium hydroxide (0.23 N) solution in refluxing systems, followed by oven drying and muffle furnace incineration (AOAC 1995) 8 .

Extraction of essential oil
The seed coats were thoroughly washed twice with distilled water, ground in electronic grinder were subjected to hydrodistillation for 4 h in Cleavenger apparatus.The essential oil was separated and collected in a sterilized glass vial.Water traces from the essential oil were removed by adding anhydrous sodium sulphate.The essential oil was stored at 4 o C in dark till used.

Determination of total phenol content
Total phenol content of the essential o i l o f T i mu r s e e d c o a t wa s d e t e r m i n e d spectrophotometrically using Folin-Ciocalteu reagent (Gholivand et al. 2010)  9 .A solution (0.1 ml) containing the 1000 µg oil was mixed with 46 ml of distilled water in a volumetric flask. 1 ml Folin-Ciocalteu reagent was added and the mixture was thoroughly shaken using electronic shaker and left for 3 min to allow completing reaction.3 ml aqueous solution of 2% Na 2 Co 3 was then added.The solution was incubated for 4 h at room temperature.At the end of the incubation period, the absorbance of each mixture was measured at 760 nm.The same procedure was also applied to standard solutions of gallic acid (0-1000µg/0.1ml)and a standard curve was obtained.
Total phenolic content of the oil was obtained by comparing the absorbance value of oil at 760nm with standard curve and expressed as µg gallic acid equivalent/ mg of oil.Absorbance = 0.0011 x gallic acid (µg) + 0.13

Antioxidant activity of Timur essential oil
Free radical scavenging activity of the essential oil of timur was measured by recording the extent of bleaching of a DPPH solution is mixed with a solution from purple to yellow (Prakash et al. 2011) 10 .During the DPPH radical assay, DPPH solution is mixed with a substance that has a capacity to donate the hydrogen atom giving rise to reduced form 1,1 diphenyl-2-picrylhydrazine (non radical).Different concentration of essential oil was added to 0.004% DPPH solution in methanol (5ml).After 30 min incubation at room temperature, the absorbance was measured against a blank at 517nm using spectrophotometer.Butylated hydroxytoulene (BHT) and Butylated hydroxyanisole (BHA) (2.0 -10 µg/ml) were used as positive controls.Scavenging of DPPH free radical with reduction in absorbance of the sample was taken as a measure of the antioxidant activity.The IC 50 of the test positive control, which represented the concentration that caused 50% neutralization of DPPH radicals, was calculated from the graph plotting percentage inhibition against concentration, while the test sample was represented as percent inhibition.I% = (A blank -A sample /A blank ) x 100

Preparation of food product from Timur seed coat powder (TSCP)
Four selected Indian food products i.e. lentil soup, chilli paneer, besan sev and Punjabi chole were developed in the laboratory.Different amounts (100-500 mg) of TSCP were incorporated in lentil soup, chilli paneer, besan sev and Punjabi chole where as standard recipe served as control.Standardization of TSCP at different levels (100-500mg) in products was done to obtain reproducible results.A group of 10 panel members were selected by threshold test (Swaminathan 1987)  11 .Acceptability test was conducted using 9-point Hedonic scale.A score card was developed to assess sensory characteristics such as colour, flavour, appearance, texture, taste and overall acceptability of the food products.Panel members were asked to assign a score using 9 point hedonic scale provided in the score card.

Lentil Soup
Lentil soup refers to a variety of vegetarian and meat soups made with lentils.The soup may consist of green, brown, red, yellow or black lentils, with or without the husk.Dehulled yellow and red lentils disintegrate in cooking, making a thick soup.In the present investigation a vegetarian lentil soup was prepared with the addition of TSCP and varying levels of TSCP to be added in per serving of soup.Ingredients for lentil soup included 15g Lentil dhal, 50g tomato, 10g onion, 3g salt, 1g turmeric powder, 5g butter, 1 bay leaf, 4 no.black pepper and 100-500mg TSCP.

Chilli Paneer
Chilli Paneer Recipe is a classic Indo Chinese dish that is inspired with the Asian sauces using Indian ingredients.It is a very popular appetizer in restaurants and parties and very easy to make as well.It can serve with other Indo Chinese Dishes like a Chilli Coriander Fried Rice.Different concentration of TSCP was standardized in per serving of Chilli paneer.Ingredients included 30g paneer, 30g corn flour, oil for frying, 20g onion, 5g ginger, 4 cloves garlic, 25g capsicum, 1 green chilli, 100mg -500mg TSCP.

Besan Sev
Besan sev, a variety of farsan or crunchy fried snacks made from Bengal gram flour.It is commonly used of snack found in every household.

Punjabi chole
Punjabi chole also known as chole masala or channay is a popular Punjabi dish in Indian and Pakisthani cuisine.The main ingredient is chickpeas.It is fairly dry and spicy with a sour citrus taste.It is often eaten with a type of fried bread and is known as bhature as well as kulcha and poori.The different concentration of TSCP was standardized in per serving chole.Ingredients used for Punjabi chole was 30g chickpeas, 50g tomato, 30g onion, 4 cloves garlic, 3g ginger, 5g salt, 3g coriander seeds, 2g cumin seeds, 4 black pepper, 1 cloves, 1 cinnamon, 1g turmeric powder, 3g red chilli powder and 100-500 TSCP.

Statistical Analysis
Experimental results were statistically analysed by mean + standard deviation of three parallel measurements.Significant differences between means were determined by paired t-test using a Microsoft Excel (Microsoft Office-2007, India) statistical analysis program and p < 0.01 was considered as significant.

Proximate Composition
The seed and seed coat was separated out from whole Timur (50g) to know the proportion of seed and seed coat.The values obtained for seeds were 22.993g (46%) and seed coats were 26.986g (54%).Table 1 showed proximate composition of whole Timur (T), seed (S) and seed coat (SC).The data indicate that moisture content of T, S and SC was 12.6%, 10.7% and 17.8% respectively.Fat content of T was 11.3 % while S and SC contained 19.5% and 6.90%.The data showed that there was a wide variation in fat content of whole Timur when compared to its separated parts i.e. seed and seed coat.Protein content was found to be 2.9% in T, 2.1% in S and 2.6% in SC.The result indicates that protein content was higher in SC as compared to T and S. The values for ash content of T and SC was similar i.e. 7.0% and 7.1% however it was slightly higher in S (7.4%).Fibre content of T, S and SC was 1.4%, 0.6% and 2.3% respectively.

Extraction of essential oil
The essential oil of seed coat of Timur was light yellow in colour and its yield was 3.5% (V/W) during hydro-distillation.Seeds of Zanthoxylum armatum DC, yielded 1.2% (W/W) of the essential oil on hydrodistillation.The yield or variation of composition of essential oil obtained from the same species may have resulted from the environmental, developmental, genetic or some other differences.Production conditions, variety, cultivars or population were some factors that also affect the yield and composition of essential oil (Singh 2003)  12 .
The standard curve of gallic acid for the measurement of total phenolic content showed in fig. 1.The total phenolic content (TPC) of the essential oil obtained by comparing the absorbance value of oil at 760nm to the standard curve was found to be 28.91+0.56µg/mg.The high phenolic content of the oil may be responsible for the higher antioxidant activity as scanvenger of the free radicals.Phenolic compounds are also very important plant constituents because their hydroxyl group confers scavenging ability (Dorman et al. 2003  13 ; Wattenberg et al. 1989 14 ; Zheng et al. 1992 15 ).

Antioxidant activity
The free radical scavenging investigations measured by recording the extent of bleaching of DPPH from purple to yellow, showed that IC 50 values of synthetic antioxidants used as positive controls was 9.7 µg/ml for BHT and 6.8 µg/ml for BHA whereas Timur essential oil has strong antioxidant activity at 100µl and 200µl showed 67.81% and 80.92% scavenging activity respectively.Essential oil of Z. alatum at a concentration of (20µg/ml) exhibited  98.5% of antioxidant activity which is significantly higher than the oleoresin, BHA and BHT (Singh  2003).Z. alatum with its antioxidant activity was expected to protect against OH mediated damage of deoxyribose (Batool et al. 2010) 16 .
Food product prepared from Timur seed coat powder Acceptability of four selected Indian food products i.e. lentil soup, chilli paneer, besan sev and Punjabi chole prepared with addition of 100-500 mg were evaluated by panel of judges.Results of organoleptic evaluation of all the four food products were depicted in table 2. The Paired t-test showed overall acceptability of lentil soup incorporated with timur powder was significantly increased as compared to that observed in the control samples to a level of 300mg of incorporation, beyond which it was not acceptable due to its high flavor and pungency.The Chilli paneer was prepared with addition of timur powder from 100-500mg was evaluated by panel of judges.It was found that overall scores for acceptability of Chilli paneer was significant higher (p>0.01) at 500mg of incorporation.Whereas besan sev prepared with incorporation of TSCP was highly acceptable upto level of 400mg.However, scores for overall acceptability were highly significant at 500mg of incorporation of timur powder in Punjabi chole which is indicative of the association between the two.Bhotiya tribal community of Uttarakhand uses timur fruit in the form of condiments, spices and medicine.They used timur as a flavouring agent or spice in certain traditional dishes.Soup made from the dried fruit (known as hag) is consumed by the entire family to keep warm during winter.A chutney (like a sauce), locally known as dunkcha, is also a popular food item in this community (Kala et al.  2005)  17 .In Nepal, timur is used in the popular foods momo, thukpa, chow mein, chicken chilli and other meat dishes.It is also widely used in homemade pickles.
Along with food uses, timur have several medicinal properties.It is used in curing various common ailments such as toothache, common cold, cough, and fever, as it is believed to give warmth to the body.Fresh or dry fruit is pressed over the affected tooth and is kept in position till it loses its pungency helps in curing decay.Recently people have also started to use powder made from the dried fruit for cleaning teeth.Common stomach complaints are treated with timur soup.The fruits and seeds are employed as an aromatic tonic in dyspepsia, carminative, stomachic, anthelmintlc and expelling roundworms.The volatile oil is employed as an antidiarrehal, antiseptic, deodorant and anticataerhal.The oil has a good tenacity and is appreciated for its fixative qualities.

CONCLUSION
It can be concluded from present study that essential oil of timur had large amount of phenolic compounds, exhibits high antioxidant and free radical scavenging activities.Ground and roasted seed coat powder of timur act as a good flavouring agents and it was highly acceptable in selected Indian food products.

Fig. 1 :
Fig. 1: The standard curve of gallic acid for the measurement of total phenolic content