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The antiviral activity of Gynostemma pentaphyllum against yellow fever virus.

Introduction

Yellow fever is a pansystemic viral sepsis with viremia, fever, prostration, hepatic, renal and myocardial injury, hemorrhage, shock and high lethality. It stands apart from other viral hemorrhagic fevers in its severity of hepatic injury and the universal appearance of jaundice (Monath, 2001, 2008). It is caused by the yellow fever virus (YFV) that is transmitted to humans through the bite of the Aedes or Haemagogus mosquitoes (Lindenbach 2007). YFV, belonging to the Flavivirus genus, is a single stranded RNA genome virus that possesses a spherical nucleocapsid surrounded by a lipid envelop in which the envelope (E) protein and membrane (M) protein are embedded. Binding of YFV to the cell surface is believed to be mediated by the E protein (Lindenbach 2007). The disease is endemic in tropical regions of Africa and South America; nearly 90% of yellow fever cases and deaths occur in Africa (Tolle 2009).

Yellow fever (YF) is a disease of significant public health importance with an estimated 200 000 cases and 30 000 deaths annually (PAHO 2004). Prevention of the disease has been by the usage of a safe and efficacious life attenuated vaccine (the 17D vaccine) but in a few vaccinees (1:200 000 to 1:300 000) the virus can cause adverse effects such as YEL-AVD (yellow fever vaccine-associated viscerotropic disease) and YEL-AND (yellow fever vaccine-associated neurotropic disease) leading to meningoencephalitis or an acute viral hemorrhagic syndrome with multiple organ system failure. The fatality rate in cases suffering from disease due to 17D vaccination is 60% (Monath 2005, Barrett 2009). There is no currently approved antiviral drug against YF and since the 1980s the number of cases of yellow fever has been increasing, making it a reemerging disease (Barrett 2007).

Effective antiviral therapeutic agents are sought to combat viral diseases in such a manner that will be toxic to the virus with minimal or no toxicity to the host cells. Unfortunately most antiviral agents have long term side effects and viruses also have the ability to adapt, mutate or negate the effect of certain antiviral agents. Currently medicinal plants are being increasingly projected as suitable alternative sources of antiviral agents because of their multiple targets, minor side effects, low potentials to cause resistance and low costs (Esimone 2010).Within the last decade the search for antiviral agents from medicinal plants against viral pandemics, which have long been a threat to human health, has been dramatically intensified (Esimone 2007, Saddi 2007, Rocio 2009, Liu 2007).

This need for new sources of lead drugs for the development of potent antiviral agents has led to the present research. The plant used in this study, Gynostemma pentaphyllum, has been claimed in complementary medicine to possess antiviral activities. It has also been reported to have various activities such as a cholesterol reducing effect (Huang 2005) and hypoglycemic effects (Samer 2006). Locally the traditional medicine practitioners use it for the treatment of bacterial and viral infections. These claims led to the screening of this medicinal plant for its antiviral activity against yellow fever virus which is a major public health concern.

Materials and methods

Collection and extraction of plant materials

The leaves of Gynostemma pentaphyllum (GP) plant were collected from Nibo in Awka south LGA, Anambra State, Nigeria. The leaves were identified by Prof CC Okeke of the Department of Botany, Nnamdi Azikiwe University, Awka. They were oven dried at 50[degrees]C for 24 h and ground to powder using a mechanical grinder.

Forty gram (40 g) portion of the plant powder was macerated in 400 mL of distilled water in a conical flask and left at room temperature for 24 hours. For the methanolic and petroleum ether extract, the 40 g portion of plant powder was macerated in 200 mL of either methanol or petroleum ether and left at room temperature for 48 hours. These were filtered using Whatman No 1 filter paper. The filtrates were concentrated to dryness in the oven at 50[degrees]C.

Phytochemical analysis of plant extracts

The extracts were first reconstituted in the respective solvents used for their extraction and then tested by standard phytochemical methods (Harborne 1998) for presence of alkaloids, flavonoids, tannins, saponins, glycosides, protein, carbohydrate, terpenoids, resins, fats and oil, acidity, steroids and reducing sugar.

Collection of embryonated chicken eggs and Swiss albino mice

Pre-incubated 9 day old embryonated chicken eggs were collected from Arroma farms Nig Ltd, Awka, Anambra State. Adult Swiss albino mice (males and females) weighing between 12 g and 18 g were collected from the National Veterinary Research Institute, Vom, Nigeria. The albino mice were fed with chicken mash and tap water. They were reared until the females became pregnant and gave birth to their litters. These suckling mice 3-7 days old were used for the antiviral assay.

Collection and preparation of the viral inoculum

Yellow fever virus already passaged in mice was obtained from the virology laboratory, University College Hospital (UCH) Ibadan and transported back to Awka in an ice pack. The virus was harvested from the brain cells of the mice using a 5 mL syringe with 21 gauge needle. The virus was filtered using Millipore micro filter of 0.45[micro]m pore size and stored in cryo vials at -20[degrees]C until used. For the egg assay, one cryo vial of the virus stock was repassaged twice in nine day old embryonated chicken eggs, harvested and stored in cryo vials at -20[degrees]C until used.

Determination of the 50% embryo infectious dose ([EID.sub.50]) and 50% embryo lethal dose ([ELD.sub.50])

Stepwise 10 fold (1/10) dilutions of the virus suspensions were made up to [10.sup.-5] in sterile test tubes using phosphate buffered saline (PBS). Five embryonated eggs were inoculated with 0.1 mL of each dilution using separate syringes and needles. The eggs were incubated at 37[degrees]C for 4 days. They were turned at least four times a day and candled each day to see if any of them had died. The dead eggs were removed from the incubator, chilled and harvested and used for hemagglutination assay. At the end of the 4 days the remaining eggs were chilled overnight and harvested and also quantified by hemagglutination assay. The result of the hemagglutination assay was used to determine the infectivity titre while the number of dead eggs and living ones were used to determine the lethal dose. These calculations were done using the mathematical technique of Reed and Muench (1938).

Determination of the 50% mice lethal dose

Stepwise ten-fold (1/10) dilutions of the virus suspension were made up to [10.sup.-5] in sterile test tubes using phosphate buffered saline (PBS). Five suckling mice less than one week old were inoculated intracerebrally with 0.01 mL of each dilution using a separate needle and syringe for each. A total of twenty five (25) suckling mice were used for the titration. The mice were observed seven days for symptoms of encephalitis and death. At the end of the seven days the number dead were used to calculate the lethal dose using Reed and Muench technique (1938).

Assay of antiviral activity against yellow fever

The assay of antiviral activity against yellow fever was determined by inoculating nine day old embryonated egg and albino mice.

Egg inoculation: The principle and procedure applied in preparation of the inoculum and egg inoculation were as described by Hawkes (1976) and Senne (1998). The nine day old embryonated chicken eggs to be used for the test were checked for viability by candling and inoculated through the blunt ends using 2 mL syringe and 21 guage needle. The eggs were inoculated with 100 [EID.sub.50] of the virus and extracts as follows: five eggs were inoculated for each concentration of extracts (200 mg/mL, 20 mg/ mL and 2 mg/mL), five eggs for each inoculation regimen (1 h pre inoculation (P1), at inoculation (0 h) and 1 h post inoculation (P0) and five eggs for each extract (3 extracts).

About 0.1 mL of the passaged virus and 0.1 mL of sterile PBS were inoculated into 5 eggs. This served as positive control while 2 mL of PBS alone inoculated into 5 eggs served as negative control. For the toxicity control 0.2 mL of the different extracts was each inoculated into 5 eggs. The eggs were incubated at a temperature of 37[degrees]C for four days. They were turned at least four times daily and also candled daily to check for egg mortality. The number of dead eggs and those alive were used to calculate the percentage protection given to the embryonated eggs by the extracts. At the end of four days the eggs were chilled overnight at 4[degrees]C and the allantoic fluid harvested and used for hemagglutination test. The result of the hemagglutination test was used to calculate the percentage inhibition of the extracts.

Mice inoculation: Ten microlitres of different concentrations of methanolic extract of the plant were separately inoculated into five suckling mice less than one week old intraperitoneally. Using 1 mL syringe with 30 guage needle the mice were inoculated as follows: five mice per concentration (20 mg/mL, 2 mg/mL and 0.2 mg/ mL), five mice per inoculation regimen (1 h pre infection, at infection and post infection). For the pre infection 0.01 mL of each extract was inoculated intraperitoneally into the mice and the mice left for 1 h before introducing 0.01 mL of 100[MLD.sub.50] of the passaged virus. For at infection 0.01 mL of 100[MLD.sub.50] of the virus and 0.01 mL of the extract were inoculated at the same time. For the post infection 0.01 mL of 100[MLD.sub.50] of the virus was inoculated into the mice 1 h before the introduction of the extract.

Positive control was 0.01 mL of virus and 0.01 mL of PBS inoculated into five suckling mice while negative control was 0.02 mL of PBS inoculated into the mice.

The mice were observed for symptoms of encephalitis (paralysis) and death for seven days. The number of mice alive was used to calculate the percentage protection given to the mice by the extracts.

Results

The phytochemical analysis of the crude extracts of the plant Gynostemma pentaphyllum (Table 1) showed the presence of saponins, alkaloids, glycosides, tannins, carbohydrates, flavonoids, resins, acidic compounds, proteins, reducing sugar and fats and oil.

Table 2 indicates the percentage protection of mice against yellow fever virus using methanolic extract of Gynostemma pentaphyllum. All the concentrations of the extracts inoculated at both pre infection and at infection gave plausible protection of 100% to the mice. The 0.2 mg/mL inoculated one hour after the virus gave the least protection of 60% to the suckling mice. In the positive control all the mice died whereas for the negative and toxicity control none of the mice died.

The aqueous extract of Gynostemma pentaphyllum at the concentration of 200 mg/mL gave 100% protection to the embryonated eggs for the three inoculation modes (pre infection, at infection and post infection). At 20 mg/mL it gave 100%, 60% and 80% protection for pre infection, at infection and post infection respectively. The 2 mg/mL concentration gave 20%, 80% and 20% protection for pre infection, at infection and post infection respectively. The positive control (virus alone) showed the death of all eggs inoculated and therefore gave 0% protection to the embryonated eggs. For the toxicity control all eggs inoculated survived showing that the extracts were not toxic to the cells (Figure 1).

The methanolic extract of G. pentaphyllum at 200 mg/mL gave 80%, 100% and 80% protection for the pre infection, at infection and post infection inoculation modes respectively. At 20 mg/mL it gave 100% protection for the three inoculation modes and at 2 mg/mL it gave 20%, 100% and 40% protection for the pre infection, at infection and post infection respectively (Figure 2).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

The ether extract of G. pentaphyllum at the concentration of 200 mg/mL gave 60%, 60% and 100% protection to embryonated eggs for the pre infection, at infection and post infection inoculation modes respectively. At 20 mg/mL it gave 100%, 80% and 100% protection for the pre infection, at infection and post infection respectively. The 2 mg/mL concentration of extract gave 100%, 60% and 80% protection for the pre infection, at infection and post infection respectively (Figure 3).

On the percentage inhibition of viral induced hemagglutination by the aqueous extract of Gynostemma pentaphyllum the 200 mg/mL (pre infection) gave the highest inhibition (85%) while the 2 mg/mL (pre infection) gave the lowest inhibition of 35%. The highest egg mortality was given by the 2 mg/mL (pre infection) and 2 mg/mL (post infection). Concentrations of 200 mg/ mL (pre infection), 20 mg/mL (pre infection), 200 mg/ mL (at infection) and 200 mg/mL (post infection) showed no egg mortality (Table 3).

The percentage inhibition of viral induced hemagglutination by the methanolic extract of Gynostemma pentaphyllum showed that the 20 mg/ mL (at infection) and 20 mg/mL post infection gave the highest inhibition of 90% while the 2 mg/mL (pre infection) gave the lowest inhibition. The highest egg mortality was given by 2 mg/mL (pre infection) while all concentrations inoculated at infection, 20 mg/mL (pre infection) and 20 mg/mL (post infection) showed no egg mortality (Table 4).

[FIGURE 3 OMITTED]

The percentage inhibition of viral induced hemagglutination by the ether extract of Gynostemma pentaphyllum showed that 200 mg/mL (post infection) gave the highest inhibition of 90% while 2 mg/mL (at infection) gave the least inhibition of 55%. The highest egg mortality was given by the 200 mg/mL (pre infection), 200 mg/mL (at infection) and the 2 mg/mL (at infection). The positive control showed all the eggs dead while the negative control showed that none of the eggs died. The toxicity control at 200 mg/mL showed that two of the eggs died indicating that there was minimal toxicity of the ether extract of GP on the embryonated eggs at 200 mg/mL. This may also be the reason for death of some eggs at the same concentration for both pre infection and at infection regimen (Table 5).

Discussion

The phytochemical screening of the extracts of Gynostemma pentaphyllum showed the presence of saponins, alkaloids, glycosides, tannins, flavonoids, carbohydrates, reducing sugar, resins and proteins. The presence of saponins, glycosides and flavonoids in the extracts of Gynostemma is consistent with other findings about the leaves of the plant (Xin 2004, Cui 1999) The leaves of Gynostemma pentaphyllum have been shown to contain more than 90 saponins and more than 100 dammarane type glycosides have been isolated and identified from it (Zhang 1993, Cui 1999).

The extracts have shown varying degrees of antiviral activities against the yellow fever virus assayed. The antiviral activities may be attributed to the rich phytochemicals contained in the extracts since various studies have shown that phytochemicals such as tannins found in almost all plant parts cure or prevent a variety of viral infections (Serafini 1994, Nonaka 1990).

Flavonoids on the other hand have been shown to exhibit inhibitory effects against viruses including HIV and respiratory syncytial virus (Li 2000). Plant polysaccharides have also been shown to exhibit potent antiviral activities especially against enveloped viruses (Hosoya 1991, Premanathan 1990). In fact Abram et al (1993) attributed the medicinal properties of Gynostemma pentaphyllum to be mainly due to the presence of saponins in the plant.

The antiviral screening of the extracts against yellow fever virus was demonstrated using three different methods: (a) protection of mice against viral infectivity by the extracts, (b) protection of chicken egg embryo against viral infectivity by the extracts, and (c) percentage inhibition of viral induced heamagglutination by the extracts.

The mice inoculation assay for the determination of the extracts' protection of mice against viral infectivity showed that the extracts were able to prevent the symptoms of encephalitis and death in many of the mice and some of the extracts gave up to 100% protection to the mice. This result corroborates other research findings that showed the antiviral activities of this plant using animal models against such viruses as Epstein-Barr virus (EBV), Herpes Simplex virus (HSV-1) and HIV-AIDS virus (Lipipum 2003, Abram 1993). Nevertheless this is the first report of the antiviral activity of this plant against yellow fever virus.

The result of the percentage protection of the embryonated eggs against viral infectivity also proved that the extracts were able to give up to 100% protection to the embryonated eggs and hence prevented mortality due to viral infection.

The result of the percentage inhibition of the replication of yellow fever virus by the extracts in embryonated chicken eggs showed that the extracts gave potent activity against the virus. Some of the extracts gave plausible percentage inhibitions of up to 90%. The result of the embryo mortality clearly showed that the extracts were not toxic to the chicken embryonated eggs since all embryos used for toxicity control survived by the fifth day of the experiment. The extracts with antiviral effect showed activity in two subsequent dilutions of the maximum non toxic concentration. This is in line with the suggestion made by Vanden et al (1993) that the antiviral activity of crude plant extracts should be detectable in at least two subsequent dilutions of the maximum non toxic concentration to ensure that the activity is not directly correlated with the toxicity of the extracts.

It is interesting to note that the extracts had activity against the yellow fever virus at different times of inoculation (one hour pre infection, 0 h at infection and one hour post infection). The extracts that inhibited viral replication at 1 h pre infection might have acted on the viral entry step of the replication cycle and prevented the virus from attachment and further replication inside the host. Those extracts that inhibited the yellow fever virus at zero hour might have acted on the virus before attachment by mechanism of binding on the active site of the host cell blocking the virus from attaching to the host receptors or by binding on the active site of the virus. According to Vanden et al (1986) polyphenols act principally by binding to the virus and/or the protein of the host cell membrane thus arresting adsorption of the virus. The extracts that inhibited 1 h post infection must have inhibited a post entry step of the viral replication. The extracts gave up to 100% protection with the three modes of inoculation. Viral infectivity was also inhibited up to 90% by the extracts of the plant inoculated in three different modes. This shows that the three modes gave good inhibition of viral growth and protection on the embryonated eggs and mice and that no mode of inoculation worked better than the other. This suggests that the extracts could be used as both preventive and curative therapy. It was observed that the antiviral activity decreased with a decrease in concentration as the 200 mg/mL concentration showed the highest activity while 2 mg/mL showed the least activity.

Conclusion

The extracts of the plant Gynostemma pentaphyllum used in this study have shown credible antiviral activities against the yellow fever virus and could be recommended as a potential source for a yellow fever remedy.

References

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Okoye EL (1), Ezeifeka GO (2), Esimone CO (3)

(1) Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, PMB 5025, Awka, Anambra State, Nigeria.

(2) College of Veterinary Medicine, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria.

(3) Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, PMB 5025, Awka, Anambra State, Nigeria.

Corresponding author: Dr Ebele Okoye, phone: 0803 664 3549, email: ebyemma2006@yahoo.com
Table 1: Phytochemical constituents of
Gynostemma pentaphyllumextracts

                         Type of extract

Constituents       Ether   Aqueous   Methanol

Saponins             +       +++        ++
Alkaloids          ++++     -+++        +
Glycosides           -       ++        +++
Tannins              -       +++        +
Carbohydrates        -       ++        +++
Reducing Sugar       -        -         ++
Flavonoids         ++++       -        +++
Resins              ++        -         +
Steroids             -        -         -
Terpenoids           -        -         -
Fats and Oils        +        -         -
Acidic Compounds     -       +++        -
Proteins             -        +        ++++

Key:

(-) = not present

(+) = present in small concentration

(++) = present in moderately high concentration

(+++) = present in very high concentration

(++++) = Abundantly present

Table 2: Mice protection against yellow fever by methanolic extract
of Gynostemma pentaphyllum

Inoculation      Extract        Mice        No     % Protection
regimen       concentration   mortality   living     of mice
controls         (mg/mL)        (D/N)              by extracts

Pre-              20mg           0/5       5/5         100
Infection          2mg           0/5       5/5         100
                  0.2mg          0/5       5/5         100
At-               20mg           0/5       5/5         100
Infection          2mg           0/5       5/5         100
                  0.2mg          0/5       5/5         100
Post              20mg           1/5       4/5          80
Infection          2mg           1/5       4/5          80
                  0.2mg          2/5       3/5          60
Positive            -            5/5       0/5          0
Control
Negative            -            0/5       5/5         100
Control
Toxicity          20gm           0/5       5/5         100
Control

Key: D = No of dead mice N = No of mice inoculated
MHA = Median haemagglutination titre

Table 3: Inhibition of yellow fever virus replication in chick embryo
by aqueous extract of Gynostemma pentaphyllum

Inoculation    Extract        Egg          MHA litre        %
regimen       concentra-   mortality       (Log 2       inhibition
controls         tion        (D/N)      reciprocal)
               (mg/mL)                     allantoic

Pre              200          0/5           1.8             85
infection         20          0/5           2.2             82
                  2           4/5           7.8             35
At               200          0/5           2.6             78
infection         20          2/5           4.2             65
                  2           1/5           5.0             58
Post             200          0/5           2.2             82
infection         20          1/5           2.8             77
                  2           4/5           7.4             38
Positive          -           5/5            12
control
Negative          -           0/5            0
control
Toxicity          20          0/5            0
control

Key: D = No of dead eggs N = No of eggs inoculated
MHA = Median haemagglutination titre

Table 4: Inhibition of Yellow fever virus replication in chick
embryo by methanol extract of Gynostemma pentaphyllum

Inoculation        Extract      Egg       MHA litre        %
regimen controls   concen-   mortality     (Log 2      inhibition
                   tration      D/N       reciprocal)
                   (mg/mL)                allantoic

Pre infection        200        1/5          4.0           67
                     20         0/5          3.6           70
                      2         4/5          6.8           43
At infection         200        0/5          1.4           88
                     20         0/5          1.2           90
                      2         0/5          2.0           83
Post infection       200        1/5          2.0           83
                     20         0/5          1.2           90
                      2         3/5          5.8           52
Positive control      -         5/5          12
Negative control      -         0/5           0
Toxicity control     200        0/5           0

Key: D = No of dead eggs N = No of eggs inoculated MHA = Median
haemagglutination titre

Table 5: Inhibition of Yellow fever virus replication in chick
embryo by ether extract of Gynostemma pentaphyllum

Inoculation           Extract      Egg       MHA litre        %
regimen controls      concen-   mortality     (Log 2      inhibition
                      tration     (D/N)     reciprocal)
                      (mg/mL)                allantoic

Pre infection           200        2/5          3.4           72
                        20         0/5          3.0           75
                         2         0/5          1.4           88
At infection            200        0/5          1.4           68
                        20         2/5          3.8           68
                         2         2/5          5.4           55
Post infection          200        0/5          1.2           90
                        20         0/5          2.2           82
                         2         1/5          2.8           70
Positive control         -         5/5          12
Negative control         -         0/5           0
Toxicity control        200        2/5           0

Key: D = No of dead eggs N = No of eggs inoculated MHA = Median
haemagglutination titre
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Title Annotation:Global dispensary
Author:Okoye, E.L.; Ezeifeka, G.O.; Esimone, C.O.
Publication:Australian Journal of Herbal Medicine
Article Type:Report
Geographic Code:6NIGR
Date:Dec 1, 2012
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