In vitro antiviral effect of ethanolic extracts from Azadirachta indica and Melia azedarach against goat lentivirus in colostrum and milk

Abstract

This study aimed to evaluate, in vitro, the use of leaf extracts of Azadirachta indica (A. indica) and Melia azedarach (M. azedarach) as antivirals against caprine lentivirus (CLV) in colostrum and milk of goat nannies. These were collected from eight individuals and infected with the standard strain of CLV. Samples were then subdivided into aliquots and treated with 150 µg/mL of crude extract, and with ethyl acetate and methanol fractions for 30, 60, and 90 min. Next, somatic cells from colostrum and milk were co-cultured with cells from the ovine third eyelid. After this step, viral titers of the supernatants collected from treatments with greater efficacy in co-culture were assessed. The organic ethyl acetate fractions of both plants at 90 min possibly inhibited the viral activity of CLV by up to a thousandfold in colostrum. In milk, this inhibition was up to 800 times for the respective Meliaceae. In conclusion, the ethanolic fraction of ethyl acetate from both plants demonstrated efficacy against CLV in samples from colostrum and milk when subjected to treatment, which was more effective in colostrum.

Figure 1

Estimate visualized in log10 with a reliability limit of 95% (Concentration (µg/mL)) in which can be observed the dependent variable “y” (concentration) as a function of “x” (time). The longer the concentrations of extracts of Azadirachta indica. and Melia azedarach and are in contact with Artemia Salina, the greater the toxicity level, thus reducing the number of living crustaceans in the study. CEE-AI: crude ethanolic extract—Azadirachta indica; EAF-AI: ethyl acetate fraction—Azadirachta indica; MF-AI: fraction methanol—Azadirachta indica; CEE-MA: crude ethanolic extract—Melia azedarach; EAF-MA: ethyl acetate fraction—Melia azedarach; MF-MA: fraction methanol—Melia azedarach.

Figure 2

Tabulated and graphed values of absorbance (570 nm) obtained by the 3–4,5-dimethyl-thiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) test after the action of crude extracts of Azadirachta indica and Melia azedarach and their respective fractions of ethyl acetate and methanol for 24 h, 48 h, and 72 h. (a) treatment with the extracts and their respective fractions at concentrations of 10, 100, and 1000 µg/mL within 24 h; (b) treatment with the extracts and their respective fractions at concentrations of 10, 100, and 1000 µg/mL within 48 h; (c) treatment with the extracts and their respective fractions at concentrations of 10, 100, and 1000 µg/mL within 72 h CEE-AI: crude ethanolic extract—Azadirachta indica; EAF-AI: ethyl acetate fraction—Azadirachta indica; MF-AI: fraction methanol—Azadirachta indica; CEE-MA: crude ethanolic extract—Melia azedarach; EAF-MA: ethyl acetate fraction—Melia azedarach; MF-MA: fraction methanol—Melia azedarach.

Figure 3

Co-culture with ovine third eyelid cells (OTE) and somatic cells from colostrum infected with CAEV_CO (SCC+) treated with Azadirachta indica and Melia azedarach leaf extracts for 90 min after 63 days of culture. (a) Negative culture control with OTE cells (×100 magnification). (b) Co-culture of OTE and SCC+ cells submitted to CEE-AI treatment, with syncytia (arrows) (×100 magnification). (c) Co-culture of OTE and SCC+ cells subjected to EAF-AI treatment (×100 magnification). (d) Co-culture of OTE and SCC+ cells subjected to MF-AI treatment (×100 magnification). (e) Co-culture of OTE and SCC+ cells submitted to CEE-MA treatment (×100 magnification). (f) Co-culture of OTE and SCC+ cells subjected to EAF-MA treatment (×100 magnification). (g) Co-culture of OTE and SCC+ cells subjected to MF-MA treatment (×200 magnification). (h) Co-culture of OTE and SCC+ with syncytia (arrows) (control treatment without extract) (×200 magnification). (i) Positive control of CAEV_CO-infected OTE cells with syncytia (arrows) (×200 magnification). CEE-AI: crude ethanolic extract—Azadirachta indica; EAF-AI: ethyl acetate fraction—Azadirachta indica; MF-AI: fraction methanol Azadirachta indica; CEE-MA: crude ethanolic extract—Melia azedarach; EAF-MA: ethyl acetate fraction—Melia azedarach; MF-MA: fraction methanol—Melia azedarach.

Figure 4

Co-culture with ovine third eyelid cells (OTE) and somatic cells from milk infected with CAEV_CO (SCM+) treated with Azadirachta indica and Melia azedarach leaf extracts for 90 min after 63 days of culture. (a) Negative culture control with OTE cells (×100 magnification). (b) Co-culture of OTE and SCM+ cells submitted to CEE-AI treatment, with syncytia (arrows) (×200 magnification). (c) Co-culture of OTE and SCM+ cells subjected to EAF-AI treatment (×100 magnification). (d) Co-culture of OTE and SCM + cells subjected to MF-AI treatment, with syncytia (arrows) (×100 magnification). (e) Co-culture of OTE and SCM+ cells submitted to CEE-MA treatment (×100 magnification). (f) Co-culture of OTE and SCM+ cells subjected to EAF-MA treatment (×100 magnification). (g) Co-culture of OTE and SCM+ cells subjected to MF-MA treatment (×100 magnification). (h) Co-culture of OTE and SCM+ with syncytia (arrows) (control treatment without extract) (×200 magnification). (i) Positive control of CAEVCO-infected OTE cells with syncytia (arrows) (×200 magnification). CEE-AI: crude ethanolic extract—Azadirachta indica; EAF-AI: ethyl acetate fraction—Azadirachta indica; MF-AI: fraction methanol Azadirachta indica; CEE-MA: crude ethanolic extract—Melia azedarach; EAF-MA: ethyl acetate fraction—Melia azedarach; MF-MA: fraction methanol—Melia azedarach.

Figure 5

Viral titers of milk and colostrum samples co-cultured with ovine third eyelid (OTE) cells submitted to 90 min treatment composed of ethanolic leaf extracts of Azadirachta indica and Melia azedarach. (a) Colostrum samples treated with crude extract of A. indica and M. azedarach leaves and respective ethyl acetate and methanol fractions. (b) Milk samples treated with crude extract of A. indica and M. azedarach leaves and respective ethyl acetate and methanol fractions. *TCID50/mL: tissue culture infection dose is the highest dilution that presented, at 14 days post-inoculation, syncytia in 50% of inoculated wells, C+: positive control of standard CAEV_CO strain, C + T.: positive control of treatments.

Figure 6

Tabulated values (a) and graphs (b) of somatic cell count (SCC) by the microscopic method in colostrum and goat milk samples after treatment with the crude extracts of Azadirachta. indica and Melia azedarach and the organic fractions of Ethyl Acetate and Methanol for 90 min of action. Different letters in the same column differ from each other by Tukey's test P < 0.01 CEE-AI: crude ethanolic extract—Azadirachta indica; EAF-AI: ethyl acetate fraction—Azadirachta indica; MF-AI: fraction methanol Azadirachta indica; CEE-MA: crude ethanolic extract—Melia azedarach; EAF-MA: ethyl acetate fraction—Melia azedarach; MF-MA: fraction methanol—Melia azedarach.

Figure 7

UPLC-ESI-QTOF-MS^E chromatograms of Azadirachta indica (1) and Melia azedarach (2) leaves extracts in negative ionization mode: (a) crude ethanol extract; (b) methanol fraction; (c) ethyl acetate. The numbers on the peaks are referenced in Tables 3 and 4.

Figure 8

Illustration of the experimental design adopted in the evaluation antiviral in vitro activity ethanolic extracts of Azadirachta indica and Melia azedarach against the caprine lentivirus in colostrum and milk. OTE: ovine third eyelid; SRLV: Small Ruminant Lentiviruses; WB: Western Blot; nPCR: nested-Polymerase Chain Reaction; CAEV_CO: standard viral strain; C-: Negative Control; C + T: Positive Control of Treatments; C+: Positive Control.