3.1 Ethnobotanical survey
3.1.1 Socio-demographic features
The study involved 142 informants, of whom 49.3% were female and 50.7% male (Table 2). Men were slightly more than women. It can be attributed to the fact that men are always involved in harvesting and selling of C.schweinfurthii fruit more often than women [34]. The findings concur with those of Tugume & Nyakoojo [7], who reported a higher proportion of male (61%) than women during an ethnobotanical survey on traditional use of wild edible plants in the communities adjacent to Mabira Central Forest Reserve, Uganda. The results are also similar to those of Tabuti et al. [9], who reported more men (58.9%) than women when documenting medicinal plants used for treatment of malaria by indigenous communities of Tororo district, Uganda. However, the current results are different from some ethnobotanical studies conducted in Uganda which reported more women participants than men [8, 21, 35].
The largest number of respondents were 18–40 years (55.6%). The presence of youth with traditional knowledge about C. schweinfurthii can be attached to its economic importance. The youth are involved in harvesting and selling of both C.schweinfurthii fruit and oleoresin. These findings differ from earlier ethnobotanical studies conducted in Uganda which reported a minimal number of youths with traditional knowledge about medicinal plants [8, 9, 35, 36]. The proportion of respondents in the 41–60 and 61–75 age categories was 33.8% and 7.7%, respectively. Few (2.8%) participants were above 75 years. Majority of the respondents were Basoga (59.8%), followed by Baganda (33.8%). The remaining 6.4% belonged to other tribes like Bagisu and Bagwere. The dominant population in Mayuge and Kamuli districts are Basoga while Luwero district is dominated by Baganda. Lusoga was recorded as the main language in the study (64.7%) followed by Luganda (33.8%) and other languages like English, Lugisu and Lugwere (1.4%). About 45.07% of the respondents attended up to primary school level, and 40.08% ordinary secondary level. This can be attributed to the current government compulsory free education policy at both primary and ordinary secondary levels in Uganda. Only 5.8% of the participants had attained both advanced secondary education and tertiary level). Few (8.4%) participants did not have any formal education. Majority of the respondents (85.9%) were involved in informal employment. This correlates well with their literacy level of the participants in this study. Few (11.9%) are formally employed and others (2.2%) were into both informal and formal employment.
Table 2
Socio-demographic features of participants
Feature | Frequency | Percentage (%) |
Gender | | |
Male Female | 72 | 50.7 |
70 | 49.3 |
Age | | |
Below 18 18–40 41–60 61–75 Above 75 | 0 | 0 |
79 | 55.6 |
48 | 33.8 |
11 | 7.7 |
4 | 2.8 |
Location | | |
Tribe | | |
Basoga Baganda Other | 85 | 59.8 |
48 | 33.8 |
9 | 6.4 |
District | | |
Kamuli Luwero Mayuge | 40 | 28.2 |
51 | 35.9 |
51 | 35.9 |
Language | | |
Lusoga Luganda Other | 92 | 64.7 |
48 | 33.8 |
2 | 1.4 |
Level of Education | | |
No formal education Primary level Secondary (O’Level) Secondary (A ‘Level) Tertairy | 12 | 8.4 |
64 | 45.07 |
58 | 40.8 |
4 | 2.8 |
4 | 2.8 |
Employment | | |
Formal Informal | 17 | 11.9 |
122 | 85.9 |
Both informal and formal | 3 | 2.2 |
3.1.2 Therapeutic information on Canarium schweinfurthii Engl.
The majority (95.8%) of the participants reported not having any documented traditional indigenous knowledge on the utilization of C. schweinfurthii. Only 4.2% indicated to have documented such. The most reported condition managed by administering C. schweinfurthii was diabetes followed by cough, high blood pressure, and diarrhea (Fig. 2). The results are similar to those of Tugume et al. [6], who reported C. schweinfurthii to be used in the management of cough, high blood pressure and diabetes when documenting ethnobotanical survey of medicinal plant species used by communities around Mabira Central Forest Reserve, Uganda. Okullo et al. [37], also reported cough, diarrhea, and hypertension as some of the conditions treated with C. schweinfurthii when documenting ethno-medicinal uses of selected indigenous fruit trees from the Lake Victoria basin districts in Uganda.
The frequently used preparation methods were maceration, decoction, infusion, and tincture (Fig. 3). Gumisiriza et al. [8], also indicated maceration and decoction to be the most common modes of preparation when documenting ethnomedicinal plants used for malaria treatment in Rukungiri district, western Uganda. It is important to note that the different plant parts are first crushed before preparation using the methods indicated. Crushing is a simple practice carried out using any material like stones [38], wooden motar and pestle. Crushing enhances the rate of extraction of bioactive compounds in water or any other medium used due to increase in the surface area of contact between the compounds and the solvents [39]. Maceration and infusion do not require any special equipment to be performed. Boiling carried out during decoction ensures the material is sterile before use [40] and preserves the herbal remedy prepared than when cold media is used. These could be the reasons why maceration, decoction and infusion are the most common methods of preparation identified in this study. For dosage, respondents indicated to use 2 tablespoon to a full cup.
The most common mode of administration of the herbal remedies was soft extract, liquid extract and the least being oleoresin (Fig. 4). This finding is in agreement with the maceration, decoction and infusion being the frequent methods of preparation identified in the current study.
The majority of the respondents (62%) acquired traditional knowledge on the use of C.schweinfurthii from family members, while 55.6% acquired it from the press (Fig. 5). A small (< 10%) portion of the respondents acquired such knowledge through other means, such as from community members, school, literature, and internet.
The calculated ICF 1 (Table 3), indicates that there was agreement amongst respondents about the medicinal effects of C.schweinfurthii in the communities. High IFC values are obtained when only one or a few plant species are reported to be used by a high proportion of informants to treat a particular disease [41]. Low ICF values mean many plant species are used by a high proportion of informants to treat a particular disease. It implies that there is no defined criteria of sharing information between informants in the community [9]. The high ICF for diabetes, cough, high blood pressure, and diarrhea shows the agreement among respondents on the use of C.schweinfurthii to manage the ailments as well as its importance. The high FL and UV levels of C.schweinfurthii for treating diabetes and cough indicated its outstanding preference.
Table 3
ICF, UV, and FL values for the diseases commonly treated with C.schweinfurthii in Central and Eastern Uganda.
Plant part used | Use category | Disease | FC | IFC | UV | FL % |
Fruit Leaves Bark of stem | Metabolic disorders | Diabetes | 106 | 1 | 0.75 | 75% |
Fruit Leaves Bark of stem | Respiratory system disorders | Cough | 73 | 1 | 0.51 | 51% |
Fruit Leaves Bark of stem | Cardiovascular disorders | High blood pressure | 34 | 1 | 0.24 | 24% |
Fruit Leaves Bark of stem | Gastrointestinal/digestive disorders | Diarrhea | 23 | 1 | 0.16 | 16% |
Fruit Leaves Bark of stem | Others | Others | 10 | 1 | 0.07 | 7% |
FC = Frequency, IFC = Informant Consensus Factor, UV = Use value, and FL = Fidelity Level |
3.1.3 Information on Canarium schweinfurthii Engl.
In Kamuli, Luwero, and Mayuge districts of Uganda, the common uses of C. schweinfurthii included food, medicine, oleoresin and others (wind breaker, charcoal, and timber). Similar findings have been reported by other researchers [1–2]. As a means of conservation, the majority of the respondents (64%) indicated use of C.schweinfurthii seeds as the form of propagation around their homestead and gardens. Respondents indicated that a seed takes 6 months – 1 year to germinate. A few (9.2%) used seedlings growing around the parent plant. Others (26.8%) depended on other forms of propagation (Table 4). The results of this study are similar to those of Mukasa et al. [36], who reported (63.6%) of respondents to have planted some medicinal plants around their homestead and gardens when documenting medicinal plants used in the traditional management of dog bites by herbalists in Eastern, Western, and Central Uganda. To ensure conservation and sustainability of wild medicinal plants, domestication and propagation strategies should be adopted and encouraged [42]. After germination, the tree takes 10 years for initial flowering and 9 months for the fruits to ripen. Participants indicated that the main maturity index for the fruit is the colour change from green to purple. The colour change can be attributed to the masking of chlorophyll pigments by anthocyanins during ripening. There is only one harvesting season from September to December. All respondents reported that there is a correlation between the age of the tree and quantity of fruits harvested (Table 4). As the tree reaches maturity, the quantity increases significantly.
Table 4
Information on Canarium schweinfurthii Engl.
Feature | Frequency | Percentage (%) |
Plant propagation | | |
Seed Seedling Other | 91 | 64.0 |
13 | 9.2 |
38 | 26.8 |
Relationship between the age of the tree and quantity of fruit harvested | | |
Yes No | 142 | 100 |
0 | |
Consumption of fruit pulp | | |
Yes No | 142 | 100 |
0 | 0 |
Any other alternative processing before consumption | | |
Yes No | 116 | 81.7 |
26 | 18.3 |
Changes in tree population over the last decade | | |
Yes No | 135 | 95.1 |
7 | 4.9 |
The therapeutic importance of a particular part of a plant is shown by the rate at which it is used by the local population [43]. The commonly used plant part(s) were fruits, followed by stem bark, oleoresin, and leaves (Fig. 6). The main reason for the common use of fruits is that they are abundant, edible, effortlessly obtained, and contain bioactive compounds. The common use of fruits results in a more sustainable form of utilisation that promotes plant conservation. The observed use of different plant parts could be to exploit their therapeutic ability inform of mixture and reduce or minimize the toxicity of efficacious plants [44]. Others researchers reported leaves as the most commonly used plant part for medicinal purposes [8, 9, 36]. The majority (94. 4%) of the respondents harvested the plant parts in the morning, while (38.7%) and (41.5%) collected them during mid-morning and evening, respectively. The different parts were harvested using pangas, sticks, and ropes. The harvested fresh materials were stored in cool dry places.
Majority (91.5%) of the respondents indicated that the plant is used for food. Respondents (80.3%) noted the plant is also used for therapeutic purposes. This correlates with the earlier finding about the various ailments (diabetes, cough, high blood pressure, and diarrhea) which are managed by administering the different concoctions from the plant. Other respondents (73.9%) use the plant for other purposes like wood and timber fetching. Participants (71.1%) use oleoresin for fragrance purposes. All respondents are consumers of C.schweinfurthii fruit. This correlates with the earlier finding of the fruit as the most commonly used part the tree. Most respondents (55.6%) reported carrying out primary processing including boiling, pounding/milling, and mixing of the plant material before use. Respondents (45.4%) did not carry out any form of processing. Most respondents (81.7%) use other traditional processing methods, which includes sun drying. Few respondents (18.3%) use cooking as the only traditional method of preparing the harvested plant materials. The fruit is blanched in lukewarm water for about 15 min to soften the pulp and the seed is pressed for oil. The bark is dried, pounded into powder which is used for medicine. The fruit can be kept for 1–2 months before treatment and only 2 days if prepared using the mentioned methods. Majority of the respondents (95.1%) noticed an increase in C.schweinfurthii Engl. tree population over the last decade. It can be attributed to re-planting done especially in Kamuli and Mayuge districts using various propagation methods mentioned.
3.2 Total Polyphenol Content, Antioxidant capacity and Cytotoxicity of C. schweinfurthii fruit pulp
3.2.1 Total Polyphenol Content (TPC)
The TPC values were calculated using the calibration curve shown in Fig. 7 and expressed as mg Gallic acid equivalents (GAE) per gram of sample in dry weight. The TPC value of the C.schweinfurthii fruit pulp was 2.48 ± 0.10 and 2.88 ± 0.02 mg GAE/g dry weight for cold and hot extraction, respectively shown in Table 5. There was no significant difference (p > 0.05) in the TPC values of C.schweinfurthii fruit pulp following cold and hot extraction methods.
Table 5
Total phenol content of Canarium schweinfurthii Engl. fruit pulp.
Method of extraction | TPC (mg GAE/g dry weight) |
Cold Extraction | 2.48 ± 0.10a |
Hot extraction | 2.88 ± 0.02a |
aMean values are not significantly different (p > 0.05) |
The TPC value of the C.schweinfurthii fruit pulp was 2.48 ± 0.10 mg GAE/g dry weight for cold extraction and 2.88 ± 0.02 mg GAE/g dry weight for hot extraction. These results were higher than 0.4808 mg GAE/g dry weight, which was reported by [45], who used fresh fruits cultivated in Nigeria whereas in the current study, the fruits were grown in Uganda. The difference in TPC may be attributed to extrinsic factors, notably (differences in soils, seasons, and climate which have been reported to affect the phytochemical yield of plant organs) as well as the duration, solvent and method of extraction [46]. Phenolic compounds are characterized as antioxidants and are capable of scavenging free radicals. The reported results for anti-oxidant capacity in this study can be attributed the total phenol content of the C.schweinfurthii fruit pulp. Therefore, the TPC of C.schweinfurthii fruit pulp may contribute to its antioxidant properties hence its therapeutic value.
3.2.2 Anti-oxidant capacity
The anti-oxidant capacity of the C.schweinfurthii fruit pulp against ROS induced in Caco-2 cell lines was studied. Reactive oxygen species in Caco-2 cell lines were induced by 2,2′-Azobis (2‐amidinopropane) dihydrochloride (AAPH). The probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) was used to determine the effect of the fruit pulp on ROS generation in Caco-2 cell lines. There was no significant antioxidant activity observed after treatment with any tested fruit concentration. As very high concentrations of extracts were tested, and higher concentrations already have no cytotoxic effect, it is assumed that the extract has low antioxidant capacity which is shown in Fig. 8. These results correlate to TPC of the sample.
The anti-oxidant activity of the fruit pulp relates to its total polyphenol content [47]. Low antioxidant capacity correlate to the low TPC of the sample. Due to their structure, polyphenols can donate electrons to reactive radicals generated by AAPH [48]. It is hypothesized that oxidation of H2DCFDA to 2′,7′-dichlorofluorescin diacetate (DCF) enhances the detection of total ROS including hydroxyl (•OH) and nitrogen dioxide (•NO2) radicals [49]. First, H2DCFDA is absorbed by cells where cellular esterase cleaves off the acetyl groups, resulting in 2,7-dichlorodihydrofluorescein (H2DCF). Oxidation of H2DCF by ROS converts the molecule to DCF, which emits green fluorescence at an excitation wavelength of 485 nm and an emission wavelength of 520 nm [49]. Another Study investigated the antioxidant activity of C.schweinfurthii fruit pulp extract using various in vitro models [50]. The results obtained in this study contrast those reported by Ayoade et al. (2015) [50], who indicated that the Canarium schweinfurthii bursaraseae seed had appreciable quantities of antioxidants in methanolic and aqueous extracts. The reported results for anti-oxidant capacity in this study can be attributed the total polyphenol content of the C.schweinfurthii fruit pulp. Polyphenols are known potent antioxidant, protecting against various cell injuries through acting as a detoxification agent, inhibiting proliferation, restoring chemo-sensitivity and exhibiting anti-neuroblastoma [51]. Furthermore, polyphenols are reported to have redox proper-ties, which are responsible for their antioxidant property thus helpful in preventing the progress of various oxidative damages (antioxidant activity).
3.2.3 Cytotoxicity
The cytotoxicity of the C.schweinfurthii fruit pulp was determined in vivo. Herein, we report for the first time the cytotoxicity of C.schweinfurthii fruit pulp from Uganda. Above pH 6.5, resazurin stained the cell culture medium purple. Resazurin reduction occurred in viable, untreated cells (control). The colour of the cell cultures was measured using a microplate reader. At 590 nm, high optical density (pink) depicted viable cells whereas low optical density (purple) values indicated cytotoxicity. The metabolism of the cells was monitored for 24 h. The 24 h values were chosen for the calculation of the lc50 because untreated cells reached maximum pink colour intensity at this time point. It was observed that C. schweinfurthii fruit pulp extract at concentrations of 1:1000–1:10 had no significant effect on cell viability and dilutions beginning from 1:5 have a significant cytotoxic effect on Caco-2 cells as shown in Fig. 9. The highest cytotoxicity (40%) was noted in Caco- 2 cell lines treated with the fruit pulp extract of 1:2 concentration. The other fruit pulp extract serial dilutions did not show any appreciable cytotoxicity since the treated Caco-2 cell lines remained viable (above 80%).
Cytotoxicity assays are the main tests used to ascertain the safety of plant extracts for human use [52]. Because most plants are believed to be safe, toxic effects of many plants are not well documented [53]. The resazurin assay assesses the survival and multiplication of mammalian cells upon exposure to plant extract(s). Above pH 6.5, resazurin stained the cell culture medium purple. Upon chemical reduction, the colour of the cell culture medium changes from purple to pink depicting viable cells, whereas purple colour indicates cytotoxicity. For a plant extract to be considered cytotoxic, it must demonstrate more than 50% resazurin death [52]. Except for concentration of 1:2, the results of the resazurin assay on the C.schweinfurthii fruit pulp showed no cytotoxic effects implying that the fruit pulp can be used for therapeutic purposes.