M. L. Sukhadiya · N. S. Thakur · V. R. Patel ·R. P. Gunaga · V. B. Kharadi · K. K. Tyagi ·Susheel Singh

Abstract This research was carried out on nine provenances from the north Western Ghats to quantify proximate principles, mineral matter, total phenols and other phytochemicals in Melia dubia drupe pulp. Drupe biometric, biomass attributes, proximate principles, mineral matter and total phenols differed significantly among the provenances. The average drupe length and width was 26.5 and 22.3 mm, respectively. Average drupe and pulp fresh weight, pulp dry weight and stone weight were 783.1, 552.1,165.1, and 179.8 g/100 drupes, respectively. Pulp, dry matter and moisture per cent were 70.5%, 29.9% and 70.0%,respectively. Average crude fibre, protein, ether extract, total ash and nitrogen- free extract were 8.3%, 7.6%, 5.1%, 6.3%,and 72.6%, respectively. Average Ca, Mg, P and K levels were 0.5%, 0.2%, 0.2%, and 2.0%, respectively. Total phenols were 0.4%. Cluster analysis showed that the study area may be divided into three distinct provenances, each having different drupe traits and proximate composition, including mineral components. Gas-chromatography mass-spectrometry analysis revealed 27 phytochemicals, many of which possess benef icial biological attributes. The implications of this provenance variation study should be useful for future improvement in terms of better drupe pulp nutrition for livestock feed. Further study suggests that M. dubia pulp could be categorized as a good feed source with low fibre, energy,fat and mineral levels. All proximate parameters, mineral matter content and total phenols are within prescribed limits.

Keywords Melia dubia · Drupe pulp · Proximate principles · Minerals · Phytochemicals

Introduction

Fodder trees and shrubs provide protein and energy that keep rumen microbes active and increase their ability to digest fibre, thus allowing livestock to make use of dry season pastures (Gaikwad et al. 2017). In India, the production of fodder has not been emphasized and is estimated to be cultivated only on about 4% of the total cultivated area (Government of India 2014). By 2020, India could face an estimated shortage of 728 MT of green fodder and 157 million tons of dry fodder (National Dairy Development Board 2012).Shortages of quality feed and fodder resources have been identified as the major constraint in livestock production in developing countries, and these countries, from time to time,experience such shortages (Ben et al. 2007). Therefore, a challenge for animal nutrition scientists is to introduce and promote alternative feed resources of good nutritive value.Wild, underutilized plant resources have been recommended to bridge the gap between availability and demand for feed resources (Sansoucy 1994).

Numerous tree species have been evaluated for their leaf fodder or pod feed quality and have been promoted to reduce feed costs by replacing the concentrates (Gunasekaran et al.2014; Gebeyew et al. 2015). Besides leaves and twigs, fruit pods are also consumed by livestock. However, limited research has been carried out on the nutritional value of fruit pulp. In addition, the use of alternative feed sources adapted to long dry seasons is important for livestock production in arid areas (Gusha et al. 2015). The interest in these alternative feed ingredients is important because of the global demand for grains which has exceeded production, and the increasing competition between humans and livestock for existing food and feed material (McCalla 2009). There are lesser-known and under-utilized plants that may have potential as livestock feed.Melia dubiaCav. is one such species where their drupes may be utilized as a top feed. This is an indigenous species of the Western Ghats region of India and is also found in Bangladesh, Myanmar, Thailand, Mexico,Sri Lanka, Malaysia, Indonesia, China, Philippines and Australia (Thakur et al. 2018). It is valued for its high-quality termite and fungus-resistant timber used for furniture, agricultural implements and house construction, for pulp and fuelwood and the leaves are used as fodder (Parthiban et al.2009).M. dubiais has been planted under industrial agroforestry models and is reported to be an acceptable agroforestry species (Mohanty et al. 2017; Thakur et al. 2018), having no allelopathic influence on understory crops (Kumar et al.2017; Parmar et al. 2018).M. dubiaalso has several medicinal properties (Susheela et al. 2008). Deer, goats and cattle often feed on fallen drupes ofM. dubiain the deciduous forests of the northern Western Ghats in times when green forage is scarce.

Research on the phytochemical characterization of M.dubia leaf (Murugesan et al. 2013) and bark materials(Sathya et al. 2017) is well-documented; however, pulp proximate analysis from a feed source viewpoint has not been determined. Proximate composition refers to the six components i.e. moisture, crude protein, ether extract, crude fibre, crude ash, and nitrogen-free extracts. Furthermore, the nutritional value of the drupe pulp is also unknown. Therefore, this study estimates the proximate principle, mineral matter, total phenols and other phytochemicals from the drupe pulp ofM. dubiafor possible provenance variation and its suitability as an alternate livestock feed source.

Materials and methods

Study sites and drupe collection

This study was carried out at the institutional research farm of the College of Forestry, Navsari Agricultural University,Navsari, Gujarat in 2017-2018. Nine provenances of M.dubia were selected in areas of the northern Western Ghats.Geographical locations, edaphoclimatic conditions of these provenances and average growth attributes ofM. dubiatrees in these provenances are given in Table 1.

Three trees (a group of three trees = one replication) were selected randomly from each of the provenances, and drupes were collected in bulk February-March, 2018, brought to the laboratory and washed and cleaned. From each replication, 100 drupes were randomly drawn from each provenance for morphometric and biomass determination. In addition, replicated drupes were de-pulped for recording pulp content, proximate principles,mineral matter, total phenols and other phytochemicals.

Drupe morphometric and biomass attributes

Drupe length (from base to the tip) and width (diameter at mid-point) were recorded. Biomass attributes such as fresh weight (g/100 drupes), pulp fresh weight (g/100 drupes),stone weight (g/100 stones), pulp dry weight (g/100),moisture content% [(fresh weight ? dry weight)/fresh weight × 100)] and dry matter% (sample dry weight/sample fresh weight × 100) were recorded.

Drupe proximate principles, mineral matter and total phenols

Proximate principles of crude fibre (%), crude protein (%),ether extract (%), total ash (%) and nitrogen- free extract(%) as well as mineral matter constituents, calcium (Ca; %),phosphorus (P; %), magnesium (Mg; %) and potassium (K;%) were estimated by standard procedures (AOAC 2016).These parameters were estimated by the Vanbandhu College of Veterinary Science and Animal Husbandry, NAU, and total phenol content was determined by the Food Quality Testing Laboratory of NAU, following standard methods of biochemical analysis (Thimmaiah 1999).

Drupe pulp phytochemicals analysis

Pulp phytochemicals were detected by gas-chromatography mass spectrometry (GC-MS) following Murugesan et al.(2013) and Sathya et al. (2017). The procedure followed is:A 1-g powdered pulp sample was placed in a 15-mL centrifuge tube, 15 mL hexene acetone (1:1) added and after 72 h incubation, the homogenate was centrifuged for 20 min at 10,000 rpm and the supernatant collected in test tubes. A second 15 mL of hexene acetone (1:1) was added to the residue left in centrifuge tube, and again after 72 h of incubation, the homogenate was centrifuged for 20 min at 10,000 rpm and the supernatant collected in test tubes. The tubes were placed in TurboVap to evaporate the supernatant to dryness. The residue was dissolved in 5 mL hexene acetone (1:1).

Average growth attributes)H (cm 28.6 47.3 38.8 54.6 31.7 39.6 32.5 43.1 50.9 trees ht (m) DB Heig.2.4.0.4 20 18 13 18.7 17.2 22.4 23.2 1.2 15 24.5.0-Average ann resent study ual tem)Max.res (°C 34.7.3 32 45 tes of M. dubia trees in p.2-4 peratu Min.20.7 9.9-23 10.0 tha 12.0 4.8 24 92 inagaw 2554.0 ch= Chin) attribu ny Soils rowth (n = 10 e di um-d ee p b l a c k w i t h f e w p a t c h e s o f c o a s t a l a l l u v i a l,l a t e r i t e iver basin Black cotton and sto Sand A ve ra ge an nu al r a i n f a l l (m m)y, m average gEdapho-climatic conditions B la ck ro ck-o ut cr op s, an d s h a l l o w b l a c k, b r o w n a n d a l l u v i a l o f r e c e n t o r i g i n s of nine provenances and/district istrict istrict a; 7 = Sakalpatal; 8 = Waghai; 9 ada R Valsad south d Dang south d Narm nda; 6 = Kaprad 5″7″4″9″7″9″1″atic condition 2′ 2 21° 4 e (E) Latitude (N) Region 0′ 5 21° 46′ 321° 36′ 020° 22′ 420° 28′ 720° 1 2′ 0 20° 4° 45′ 22″″20 20° 47′ 18″pur; 5 = Nanapo raphical location, edapho-clim itud 4′ 27″3″0″9′ 5 73° 2 de (m a.s.l.) Long 73° 48′ 13″73° 47′ 79″73° 44′ 84″73° 08′ 97″73° 09′ 80″73° 9′ 36″73° 3 3′ 4= Dharam 73° 3 ra; 4 og 340.0 7.3 437.0 394.0.0 23 13 25 639.0 9.9 Geog ces Altitu 12 467.0 6.2 Table 1 Provenan H diameter at breast height 123456789D B 1 = Ingawadi; 2 = Sagai; 3 = Devm

The GC-MS analysis was carried out on with a Thermo Trace GC-ULTRA-ITQ 900 with a fused silica capillary column (Rx-1-5MS) 30 m long, 0.25 internal diameter and 0.25 μm film thickness. The GC oven was set to an initial temperature of 70 °C for 3 min and then increased at the rate of 8 °C per minute to 290 °C and kept for 4 min. The injection was performed in PTV split less mode with a split flow of 50 mL min ?1 . The temperature of the injector was 250 °C. Helium (ultra-pure 99.99%) was used as carrier gas with 1.0 mL min ?1 flow rate at a constant flow mode. The ionization voltage was 70 eV. The source temperature of mass spectrometer transfer line was kept at 230 °C. The rate of micro-scanning was 3 scan s ?1 and samples were run in full scan mode with mass range 50-650 dalton (atomic mass unit).The samples were injected with an AP/AS 3000 auto sampler with an injection volume of 1 μL a total run time of 33 min.

Statistical analysis

Data pertaining to drupe biometric and biomass attributes,proximate principles, mineral matter and total phenols were analyzed per standard statistical procedures using a complete randomized design (CRD) following Snedecor and Cochran(1980). The means of different treatments were tested for statistical significance of P ≤ 0.05 using Duncan’s multiple range tests. Non-hierarchical Euclidean cluster analysis was done for various drupe parameters using Spar-1 statistical program(Doshi and Gupta 1981). With parameters in percent, arc-sine transformation was done and used to express the results.

Results and discussion

Drupe biometric and biomass attributes

There was significant variation between drupe biometric and biomass attributes among the different provenances(Table 2). Average length and width were 26.5 and 22.3 mm,respectively. Average fresh weight, pulp fresh weight, pulp dry weight and stone weight was 783.1 g, 552.1 g, 165.1 g and 179.8 g/100 drupes, respectively. Average pulp content,dry matter, and moisture content were 70.5%, 29.9%, and 70.1%, respectively. Biometric and biomass trait variations between provenances may be attributed to the edaphoclimatic and genetic variations in individuals of different provenances (Bhagora 2017), and the provenances in this study also have varied edaphoclimatic conditions, the influence of soils on living organisms, as mentioned in the site descriptions. Moisture inM. dubiadrupes was recorded as high as 71.0% and averaged 70.0% in the provenances in this study.

Table 2 Variations in drupe biometric and biomass attributes of Melia dubia among different provenances

Drupe pulp proximate principles and total phenols

These varied significantly among the provenances (Table 3).Overall, mean crude fibre, crude protein, ether extract, total ash and nitrogen-free extract of drupe pulp was 8.3%, 7.6%,5.1%, 6.3%, and 72.6%, respectively. These variations in proximate parameters could be applied for selecting provenances for specific purposes, i.e., if the pulp is to be used to provide fat rich feed then the Waghai and Kaprada provenances may be considered as these have more ether extract.

Crude fibre from drupe pulp ranged from 7.7 to 9.2% with an average of 8.3%. Feed concentrates having crude fibre less than 18% are considered good (National Dairy Development Board 2012). Hence,M. dubiadrupe pulp could be considered a good feed source with low fibre content. In general,the higher the fibre, the lower the energy content of feed(National Dairy Development Board 2012). Crude protein ranged from 6.7 to 9.2% in drupe pulp represented by different provenances with an overall mean of 7.6%. Notably, concentrates having less than 18% crude protein are classified as energy rich feed source (National Dairy Development Board 2012). Total ash ranged between 5.6 and 7.1% with a mean of 6.3%. The relatively large amount of total ash suggests a high-value mineral composition comprising potassium, calcium and iron as the main elements (Gaikwad et al. 2017).Nitrogen-free extracts were 72.6% and ranged from 70.8 to 73.7%. Feed sources with such range are considered energy rich (Government of India 2014).

Table 3 Variations in Melia dubia drupe pulp proximate composition and total phenols among different provenances

The range of proximate principles inM. dubiadrupe pulp falls within average values reported in many common leaf and pod feed sources throughout the world (Adepoju 2009;Chandra and Mali 2014; Gebeyew et al. 2015; Gaikwad et al.2017). However, crude protein levels are even higher than in many common feed resources of many species. Wastes of Citrus spp. and jackfruit have been reported to contain 65.0-75.0% and 65.3% nitrogen-free extracts, respectively.These extracts in leaves of commonly used fodder trees and shrubs have been documented (Gunasekaran et al. 2014) and are lower than that found inM. dubiadrupe pulp. While exploring alternative feed stocks for small ruminants,Andrade-Montemayor et al. (2011) reported that, for different varieties of wild cacti, composition varied from 8 to 15%dry matter, 5-7% crude protein with 10-30% of total N represented by crude protein-acid detergent fibre. Ash content(7.2%) was highest (P< 0.001). These proximate parameter values are within the ranges found inM. dubiadrupe pulp.

Drupe pulp total phenols

The percentage of total phenols differed significantly among the nine provenances (Table 3). Overall total phenol content was 0.4%. Total phenols in important fodder and forage species ranged from 0.15 to 0.48 mg g ?1 in leaves and 0.11-0.32 mg g ?1 in pods; however, these values are not considered toxic (Caramori et al. 2004). Similar inferences have also been made by Paterson et al. (1998). Therefore,the range of total phenols inM. dubiadrupe pulp follow this range and hence may be advantageous for livestock.

Drupe pulp mineral matter

Calcium and Mg varied significantly; however, there was no statistical difference in P and K percentage among the nine provenances (Table 4). Overall average per- cent Ca, Mg, P,and K were 0.5, 0.2, 0.2, and 2.0%, respectively.

It is important to note that the levels of Ca and Mg in the fruit pulp are considered adequate, P is at the critical level and K content is higher than the minimum standard nutrient requirements of dairy cattle set out by the National Research Council, Washington, DC (National Research Council 2001). The estimates for Ca, Mg and K inM. dubiadrupe pulp in this study are within the range ofleaf fodderand pods of many tree species, for example, Acacia spp.(Bhowmik et al. 2008; Ansari et al. 2012; Abdalla et al.2014). Annatto (Bixa orellana) seed meal (an industrial byproduct), promoted as goat feed, contains Ca, Mg and K(Kumar 2007); however, Ca levels are lower compared to M.dubia pulp. K contents in forage grasses have been reported in the range of 1.0-1.2% of dry matter (Kumar and Soni 2014). In contrast,M. dubiapulp had much higher potassium levels than these forage grasses. Phosphorous contents in forage grasses as well as in the pods and leaves of many tropical and temperate fodder species (Ansari et al. 2012)fall within the range reported forM. dubiapulp in the present study. P levels in the pods of Acacia nilotica were found to be slightly higher than 0.1% (Abdalla et al. 2014), which is less than the amount in drupe pulp ofM. dubia.

Table 4 Variation in Melia dubia drupe pulp mineral matter composition among different provenances

M. dubia provenance cluster analysis

This showed that there was genetic variation among the nine provenances which formed three distant groups (Tables 5,6). Provenances of cluster 1 represent the Dangs region and are found on the Deccan Trap ([large igneous province located on the Deccan Plateau of west-central India (17°-24°N, 73°-74° E)] with medium to high rainfall (Table 1). Similarly, cluster 2 are on the northern extension of the Deccan Plateau in central India with different soil and climatic features. Cluster 3 is climatically and edaphically different than the other two regions and belongs to Narmada River basin.Therefore, overall results show three distinct provenances having different features in terms of drupe traits and proximate composition, including a few mineral components.The cluster patterns shown by geography provide the basis for genetic diversity (Zobel and Talbert 1984), and may be considered as three broad provenances. Drupes may be collected from a better provenance for the preparation livestock feed. Such studies have been described based on fruit/pod and seed traits for many species (Divakara and Das 2014;Wani et al. 2015).

Table 5 Clustering of M. dubia provenances selected in present study

Table 6 Inter and intra euclidean distance among three clusters

Other phytochemicals from drupe pulp

A total of 27 phytochemicals were identified inM. dubiadrupe pulp in nine provenances. (Table 7). Drupe pulp of provenances 2, 3, 4, and 5 had the maximum number of compounds (27) with prominent percent area under the curve (Table 8, Figs. 1, 2, 3), followed by provenances 1, 6, 7, 8, and 9. This study showed that 1-Tetradecanol in Ingawadi and Kaprada provenances; tetradecanedioic acid in Sagai and in Devmogra; Cycloheptanemethanol,α-α-dimethyl- in Dharampur; 2-Phenylanthraquinone in Nanaponda; 3-Fluoro-5-trifluoromethylbenzoic acid,2,4-dichloro-6-formylphenyl ester in Sakalpatal; propionic acid in Waghai and dimethyl 2-carbethoxy-cyclopropane-1,1-dicarboxylate, in Chinchinagawtha, were dominant based percent area under curve.

Table 7 Phytochemicals in M. dubia drupe pulp from nine provenances

Table 8 Variation in M. dubia drupe pulp phytochemicals among different provenances

The phytochemical compounds detected inM. dubiadrupe pulp have also been detected in different plants.The literature shows that many of these compounds have several benef icial properties and biological activities. Phytochemicals in Table 5, listed at sr. no. 2, 8, 15, 16, 19,20, 21, 26, and 25 have been reported to have important biological activities like analgesic, antibacterial, anticancer, anti-diabetic, antifungal, anti-inflammatory, antitumor, anti-ulcer, astringent, cathartic, diuretic, laxative etc.(Ramalakshmi and Muthuchelian 2012; Brokl et al. 2013;Chien et al. 2015; Otieno 2016; Iwara et al. 2017). Based on the literature,M. dubiadrupe pulp contains many notable phyto-chemical compounds that may be used as an alternate feed source for livestock with possible benef icial effects.

Fig. 1 Chromatograms of retention time and relative abundance of phytochemical compounds in M. dubia drupe pulp in provenances 1, 2, 3 and 4 (a- d, respectively)

Fig. 2 Chromatograms of retention time and relative abundance of phytochemical compounds in M. dubia drupe pulp in provenances 5, 6, 7 and 8 (a- d, respectively)

Fig. 3 Chromatograms of retention time and relative abundance of phytochemical compounds in M. dubia drupe pulp in provenance 9

Conclusions

The results indicate that the nutritive value of drupe pulp varied significantly among the nine provenances. Based on levels of crude fibre, crude protein, nitrogen-free extracts, ether extracts and total ash,M. dubiapulp could be regarded as good feed source with low fibre content,and high energy, fat-rich and mineral- rich. The range of phenols is within permissible limits. The results of the cluster analysis shows that the study area may be divided into three distinct provenances with different drupe traits and proximate composition, including mineral components. Gas chromatography- mass spectrometry analysis revealed as many as 27 phytochemicals, some with medicinal properties. Therefore, the drupe pulp of M.dubia is a source of these valuable phytochemicals for livestock. Provenance variation in nutritional and phytochemical attributes be considered for the selection of better provenances for livestock feed source, and for future improvement studies to enhance nutritional values and extraction of benef icial phytochemicals. In summary, the proximate principles, mineral matter and total phenol levels inM. dubiadrupe pulp are within permissible limits(recommended for livestock), and fall within the range or higher than found in leaves, twigs and pods/fruits of many commonly used fodder species. This study shows thatM. dubiadrupes are a good alternative, agro-industry by-product for livestock feed.

AcknowledgementsThe authors are thankful to Dr. B.N. Patel,Dean, ASPEE College of Horticulture and Forestry, and Dr. N.H.Kelawala, Dean, Vanbandhu College of Veterinary Sciences and Animal Husbandry, NAU, Navsari, Gujarat for providing support for this study.