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Effects of Petiveria alliacea (guinea hen weed) leaf extract on methane production and rumen fermentation in vitro

K O Adebayo, F T Olagoke, R M Akinbode and R Y Aderinboye

Department of Animal Nutrition, College of Animal Science and Livestock Production, Federal University of Agriculture, Abeokuta Nigeria


In vitro gas production study was carried out for 48 h to determine the effect of Petiveria alliacea leaf extract on methane production and rumen fermentation. P. alliacea leaf extract of varying concentrations (0, 2, 4, and 6%) was added to a substrate separately to make five treatments in a completely randomized design. Secondary metabolites in the varying concentrations were determined. At the end of the 48 h incubation period methane was estimated and dry matter digestibility was determined. P. alliacea leaf extract is high in saponin, and flavonoid. Other secondary compounds present include tannin, phenol, oxalate, phytate, trypsin inhibitor and cyanogenic glycoside. The extract reduced (P<0.05) total gas and methane gas production. The lowest percentage of methane in total gas was obtained at 4 and 6% concentration of the extract. Highest methane depression (12.3%) was obtained in the substrate incubated with 4% concentration of P. alliacea leaf extract. Dry matter digestibility decreased (P<0.05) as the concentration ofP. alliacea leaf extract in the substrate increased. P. alliacea leaf extract can be used to manipulate rumen fermentation to reduce methane production without deleterious effect on feed degradability at 4% (4 g/100ml) concentration.

Key words: digestibility, gas production, incubation, secondary metabolites


Enteric methane production by ruminants during feed fermentation in the gut contributes to increasing problem of global warming in many countries of the world due to accumulation of greenhouse gases (Bamikole et al 2019). As the world human population is increasing so also is agricultural production to meet nutrient requirement and invariably increased methane production from ruminant livestock. Methanogenesis in the rumen also represents about 12% energy loss from gross energy intake by ruminants (Kim et al 2012). Hence, the production of methane poses a great challenge to both livestock and the environment. For this reason numerous efforts are geared towards reducing methane emission from livestock (Medjekal et al 2016). Among these are a number of nutritive strategies designed to mitigate enteric methane formation by focusing on the potential addition of distinct plants or extracts rich in secondary compounds to animal feeds (Rira et al 2015). Recent studies (Bamikole et al 2019; Adebayo et al 2019; Aderinboye et al 2020) have shown that many medicinal herbs and spices rich in secondary compounds could be used to manipulate rumen fermentation favourably. Bhatta et al (2012) reported that some of the plant secondary metabolites such as tannin, glycosides, polyphenol, trypsin which act as rumen modifiers, due to the fact that they occur naturally and are thus environmentally friendly are more widely accepted when it comes to food safety. They are therefore a better alternative to antibiotics which usage has been restricted and/or out rightly banned in some countries due to safety concerns.

Petiveria alliacea is a wild and perennial shrub that grows in Africa and tropical America (Camargo 2007). P. alliacea is a very important plant in traditional Latin America herbal medicine where it is used as an anti-rheumatic, anti-inflammatory, to treat fever, headache, diabetes, malaria, arthritis, skin allergies, cancer, and to induce abortions (Pérez-Leal et al 2006). In Brazil, Cuba, and tropical Africa it is important in Yoruba magical rituals (Alegre and Clavo, 2007). João et al (2018) reported that Petiveria alliacea contained some phytochemicals (secondary metabolites) such as flavonoids, terpenoids, and benzenoids which are commonly identified in the plant. Monache et al (1996) opined that leaves of Petiveria alliacea contain flavonoids such as 6-C -formyl and 6-C-hydroxymethyl have been identified, as well as other compounds including glycosides, saponins, triterpenes, isoarborinol, isoarborinol acetate, isoarborinol cinnamate, steroids, alkaloids and tannins. The herb is rich in sulfur‑containing compounds similar to allicin found in garlic and onion (de Andrade et al 2012).). Tropical plants containing tannins/ saponins have been reported to suppress or eliminate protozoa from the rumen and reduce methane production (Patra and Saxena, 2010; Bodas et al 2012). In vitro gas production techniques are widely used to evaluate the anti- methanogenic potential of rumen fermentation modifiers such as plant secondary metabolites (Garcia- Gonzalez et al 2008). This study therefore aims at assessing the effect of Petiveria alliacea leaf extract on methane production and rumen fermentation using the in vitro method. There is a dearth of published information on the use of P. alliacea as rumen modifier or as feed resources for ruminants hence the need for this study.

Materials and methods

Experimental site

The study was carried out at the Department of Animal Nutrition laboratory, Federal University of Agriculture, Abeokuta (FUNAAB) Ogun State, Nigeria. Ogun State is in the rainforest zone of South West Nigeria. The area has an annual mean temperature of 34.7°C, a relative humidity of 82% and an annual mean rainfall of 1,037 mm. It is about 70 m above sea level and lies on latitude 7°5'-7°8'N and longitude 3°11.2'E.

Processing of Petiveria alliacea leaf extract

Petiveria alliacea Guinea hen weed) leaves was harvested in a nearby village around the University. The leaves were harvested at the petiole and air dried to constant moisture level. The dry leaf was milled to pass through 1mm sieve. Extraction was by hot infusion method; 100ml of hot water was added to 0, 2, 4 and 6 of the leaf powder in separate jars to obtain 0%, 2%, 4% and 6% concentration of extract. The solution was allowed to stand for 20 minutes and thereafter sieved with Whatmann NO.1 filter paper to obtain a clear solution. This is a modification of the method described by Imaga and Bamigbetan (2013).

Experimental substrate

The experimental substrate consist of Panicum maximum and concentrate mixture in the ratio of 6:4. The concentrate was formulated to contain approximately 14% crude protein with the following ingredients: wheat offal 30%, maize bran 27%, PKC 20%, rice bran 20%, bone meal 2% and salt 1%.

Chemical analyses

The secondary compounds in P. alliacea were determined. Tannin was determined following the procedure of Polshettiwar et al (2007), Alkaloid was determined according to Harborne (1973), Cyanogenic glycoside was determined following the procedure of Onwuka (2005), Oxalate was determined according to the method described by Munro (2000) ,Phytic acid was analysed according to Oboh et al (2002), Flavonoid was determined following the procedure of Mahajan and Badujar (2008), Phenol was determined following the procedure of Rajeev Singh et al (2012) and Saponin was determined according to Obdoni and Ochuko (2001).

Photo 1. Petiveria alliacea L
In vitro gas production study

In vitro gas production was carried out according to Menke and Steingass (1988). Two hundred milligram (200 mg) of dried and milled sample of Panicum maximum and concentrate in ratio 6:4 was weighed into 100 ml calibrated transparent glass syringes fitted with silicon tube and 0.5ml Petiveria alliacea leaf extract of five different concentrations (0, 2, 4, 6 and 8%) was added to the substrate to make five treatments which were replicated eight times. Thereafter the syringes were filled with 30 ml of medium consisting 10ml of rumen fluid and 20ml of buffer solution (NaHCO3+3Na2HPO4+KCl +NaCl+MgSO 4.7H2O+CaCl2.2H2O). The syringes were tapped and pushed upward by piston to eliminate air completely in the inoculums. The silicon tube in the syringe was tightened by a metal clip so as to prevent escape of gas. Incubation was carried out at 39oC and the volume of gas produced was measured at three hours interval from 0-48 hours. Three blanks containing 30 ml of medium only was included in the run to correct for gas production not arising from degradation of substrate. At post incubation period, 4ml of sodium hydroxide (10M) was introduced to estimate the methane production as reported by Fievez et al (2005).

In vitro dry matter digestibility

At the end of incubation, the residue from each syringe was recovered by filtering and then oven-dried at 105oC for 24hours. The dry residues were then weighed and degradability calculated as the difference between the incubated sample and residue. The difference was then expressed as a percentage of the incubated sample

Statistical analysis

Data collected were subjected to one-way analysis of variance in a completely randomized design using version 9.1 of SAS software (SAS 2003) with the following model Yij = µ + Ti + eij, Where Yij= Observed variation, µ = Population mean, Ti = effect of ith varying concentration of extract (1-5), eij = error term. Significant means were separated using Duncan’s procedure of the same software. Mean differences were considered significant at P< 0.05


Table 1 shows the secondary compounds inherent in Petiveria alliacea leaf extract at different concentrations. The presence of tannin, phenol, oxalate, phytate, trypsin inhibitor, flavonoid, cyanogenic glycoside, alkaloid and saponin were recorded in this study. Saponin was highest at the different concentrations while cyanogenic glycoside was minute.

Table 1. Secondary compounds in Petiveria alliacea leaf extract at varying concentrations





Tannin (%)




Phenol (%)




Oxalate (%)




Phytate (%)




Trypsin inhibitor (%)




Flavonoid (%)




Cyanogenic glycoside (%)




Alkaloid (%)




Saponin (%)




Presented in table 2 and Figure 1 is the total gas production from substrates containing different concentrations of P. alliacea leaf extract at 48 hours of incubation. Total Gas production was highest gas (P<0.05) in substrate containing no extract and least gas production obtained in substrates containing different concentrations of P. alliacea leaf extract.

Table 2. Effect of different concentrations of Petiveria alliacea of leaf extract on total gas production

Concentrations of Petiveria
leaf extract (%)

Total gas production
(ml/200mg DM)











p value


abc means along the same column with different superscript are different at (P<0.05)

Figure 1. Curvilinear trend between total gas production and substrates incubated
with varying concentrations of Petiveria alliacea leaf extract

Table 3 shows methane gas production (ml/ 200mg DM) and methane in total gas (%) as influenced by varying concentrations of P. alliacea leaf extract. The amount of methane produced reduced as the concentration of P. alliacea leaf extract in the substrate increased. Methane production was highest in substrates containing 0% of P. alliacea leaf extract and lowest in substrate containing 4% and 6 % P. alliacea leaf extract concentrations (Figure 2). Methane in total gas production also followed similar trend (Figure 3). Methane depression was highest (12.3%) in substrate incubated with 4% P. alliacea leaf extract.

Table 3. Effect of different concentrations of Petiveria alliacea leaf extract on methane production

Concentrations of Petiveria
leaf extract (%)

Methane production
(ml/200mg DM)

Methane in
total gas (%)
















p value



abc means along the same column with different superscript are different at (P<0.05)

Figure 2. Curvilinear trend between methane gas production (ml/200mg DM) and
substrate incubated with varying concentrations of Petiveria alliacea leaf extract
Figure 3. Curvilinear trend between methane in total gas (%) and substrate
incubated with varying concentrations of Petiveria alliacea leaf extract

Dry matter degradation decreased as the concentrations of Petiveria alliacea leaf extract increased (Table 4; figure 4). The reduction in digestibility ranged from 4 to 21% in substrate incubated with 2% and 6% P. alliacea leaf extract respectively

Table 4. Effect of different concentrations of Petiveria alliacea leaf extract on dry matter degradation

Concentrations of Petiveria
leaf extract (%)

Dry matter
degradation (%)











p value


abc means along the same column with different superscript are different at (p<0.05)

Figure 4. Curvilinear trend between dry matter degradation and substrates incubated
with varying concentrations of Petiveria alliacea leaf extract


Secondary compounds in Petiveria alliacea leaf extract include tannin, phenol, oxalate, phytate, trypsin inhibitor, flavonoid, cyanogenic glycoside, alkaloid and saponin as confirmed in this study. Previous studies by Kubec et al (2003) and de Andrade et al (2012) also revealed the presence of flavonoid and saponin in P. alliacea. Delle- Monache et al (1996) also reported that glycoside, saponin, alkaloid and tannin have been identified in P. alliacea. The presence of these compound indicated that it can be used in manipulating rumen fermentation. Rumen fermentation can be modified through the use of plant secondary metabolites (Bhatta 2015).

Gas production at 48 h incubation time is lower than what was reported by Getachew et al (2002) and and Kamalak et al (2005). P. alliacea leaf extract reduced total gas production which is an indication of reduced digestibility since the amount of gas produced depends on the quantity of substrate digested. The quantity and rate of gas produced during in vitro incubation is a true reflection of the degree of fermentation and degradability of a feed (Adeyemi et al 2015). It also suggested that secondary compounds in P. alliacea leaf extract reduced the activities of microorganisms in the rumen especially bacteria that were involved in feed degradability. According to Yusuf et al (2013) anti-nutritional factors inactivate some microorganisms in the rumen hence reduce fermentation and gas production. It has been reported that tannins reduced feed/nutrient degradation (Tavendale et al 2005; Jayanegara et al 2011). This observation corroborated the report of some authors (Iyere et al 2019; Bamikole et al 2019; Aderinboye et al 2020) who also reported that medicinal plants, herbs and spices reduced in vitro gas production. In contrast, Binh et al (2018) and Inthapanya et al (2020) reported an increase in gas production when cassava root was supplemented with cassava leaves and a range of additives. The variance is probably due to different substrates and additives that were used. The lowest volume of methane obtained at 4 and 6% concentration suggested that secondary metabolites in P. alliacea leaf extract at 4% and 6% were able to suppress methanogenesis. Several studies (Bodas et al 2008; Garcia- Gonzalenz et al 2008; Kamra et al 2008, Jahani-Azizabadi et al 2011; Bamikole et al 2019, Aderinboye et al 2020) also reported that some medicinal plants, herbs and spices decrease methane production in vitro. In addition, cassava root pulp incubated with varieties of additives and cassava leaves has also been reported to reduce methane production with “bitter” compared with “sweet” variety of cassava leaves (Binh et al 2018; Inthapanya et al 2019; Inthapanya et al 2020). These studies further affirmed the influence of secondary compounds on methane production. Sarkiyayi and Agar (2010) reported higher anti-nutritional factors (cyanogenic glycosisde, trypsin inhibitor, oxalates, phytates, and tannin) in bitter cassava leaves when compared with the sweet variety. Secondary metabolites such as saponin (Hess at al 2003; Wang et al 2009; Sirohi et al 2009; Goel and Makkar 2012), tannin (Babayemi et al 2004; Jayanegara et al 2011; Anantasook et al 2014 ), Flavonoids ( Broudiscou at al 2002; Kim et al 2015;), phenol ( Asiegbu, 1995) have been found to reduce methane production. Scehovic (1999) reported that identifying the secondary compounds specifically involved in methane reduction is difficult. Tannins have inhibitory effect on methanogens, protozoa and other hydrogen-producing microbes which enables them to reduce methane production (Patra et al 2010). The values (26.8- 39.5%) obtained for methane in total gas were in agreement with what was recorded by Bamikole et al (2019) for some medicinal plants. The high methane volume at 0 and 2% concentration is indicative of energy loss to the animal as well as increased concentration of methane in the atmosphere. A decrease in methanogenesis would lead to a major increase in the metabolizable energy content of the diet and invariably an increase in productivity of the host ruminant (Inthapanya et al 2019).

Min et al (2002) reported that tannin inhibits dry matter digestibility. The reduction in dry matter degradability as the concentration of the extract in the substrate increased is therefore indicative of the activities of secondary metabolites present in P. alliacea. Vongsamphanh et al (2018) also observed a reduction in dry matter digestibility when cassava leaves were from bitter rather than sweet variety in an in vitro rumen incubation of cassava root pulp- urea with cassava leaves. The authors also suggested that higher concentration of cyanogenic glycosides and other secondary compounds in the bitter cassava leaves were having an inhibitory effect on methanogens and the rumen microbiota in general. The reduced dry matter digestibility recorded may be responsible for the decrease observed in total gas and methane gas production. The decrease in methane gas production and dry matter digestibility obtained in this study corroborated the results of previous studies (Animut et al 2008; Tiemann et al 2008, Abdalla et al 2012; Oskoueian et al, 2013, Joch et al 2019; Aderinboye et al 2020) where methane abatement were attributed to a decrease in feed digestibility. According to Goel and Makkar (2012), methane mitigation partially is due to decrease in feed digestibility and substantial reduction in methane emission would be difficult to achieve without decreasing the feed digestibility. However, some authors (Bodas et al 2008; Kamra et al 2008; Staerfl et al 2010; Medjekal et al 2017) reported a reduction in methane gas production with no adverse effect on feed degradability and total gas production. Substrate incubated with P. alliacea leaf extract at 0, 2 and 4% concentrations have dry matter digestibility values greater than 50% which is line with the reports of Bodas et al (2008) and Medjekal et al (2017) for some medicinal plants. The highest methane depression of 12.3% obtained in this study was lower than 15% and 25 % abatement obtained by Bodas et al (2008) and Kamra et al (2008) respectively. Inthapanya et al (2019) also recorded a 50% reduction in methane when simulated rice distiller’s by-product fermented by yeast (SRDB-F) was incubated with cassava root supplemented with urea and bitter cassava leaves. The differences observed in methane reduction can be attributed to different substrates and additives used. Substrate incubated with 4% concentration of P. alliacea leaf extract depressed methane production with minimal reduction in feed digestibility. Reduction in methane production with minimal decrease in feed digestibility may be advantageous as some nutrients especially energy and protein could escape wasteful degradation by microbes and escape into the lower tract where they will be subjected to enzymatic digestion and absorption in the small intestine. No previous publication was found on the use of P. alliacea leaf extract/ meal as rumen modifier or as feed resource for ruminants.



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